Endocrine Disruptor Methods Validation Subcommittee August 2003

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2 . Relative EVALUATION OF VITELLOGENIN METHODS FOR FATHEAD MINNOW, MEDAKA AND ZEBRA FISH. WORK PERFORMED BYOn sake of the United States Environmental Protection AgencyEPA CONTRACT NUMBER 68-W-01-023. 3 . Presentation. Motivation behind study: Survey of Methods for estimation of Vitellogenin (VTG)to conduct an overview of existing VTG investigative strategies for suitability in a normal screening system.

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Endocrine Disruptor Methods Validation Subcommittee August 2003 EPA Work Assignments: 2-19 and 2-26 Presented by: Michael L. Blanton and Dr. Irv Schultz COMPARATIVE EVALUATION OF VITELLOGENIN METHODS FOR FATHEAD MINNOW, MEDAKA AND ZEBRA FISH Photos: Biosense Laboratories

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COMPARATIVE EVALUATION OF VITELLOGENIN METHODS FOR FATHEAD MINNOW, MEDAKA AND ZEBRA FISH WORK PERFORMED BY On benefit of the United States Environmental Protection Agency EPA CONTRACT NUMBER 68-W-01-023 2

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Introduction Purpose of study: Survey of Methods for estimation of Vitellogenin (VTG) to lead an overview of existing VTG diagnostic strategies for appropriateness in a normal screening program. Study was not expected to be a Method Validation the examination was not planned to be an approval of a given technique, however an assessment crosswise over strategies to learn the subjective as well as quantitative similarity of the assortment of techniques presently accessible. 3

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VTG Background VTG is a phospholipoglycoprotein antecedent to egg yolk protein that regularly happens in sexually dynamic female oviparous fishes, yet can be instigated to happen in guys in light of estrogenic substances. The estimation of a biochemical marker, VTG in oviparous vertebrates is for the most part consented to be a decent pointer for estrogenic and hostile to estrogenic impacts and is proposed as one of a few endpoints in the fish screening examine. 4

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Methods for Measuring VTG enlistment in fishes Enzyme-Linked Immunosorbant Assays (ELISA) A chemical immunoassay using a compound marked immunoreactant (antigen/counter acting agent) and an immunosorbent (antigen/neutralizer bound to a strong support – i.e, a polystyrene microliter plate) 5

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Methods for Measuring VTG acceptance in fishes cont. mRNA location An other option to measuring the VTG protein is to evaluate the errand person ribonucleic corrosive (mRNA) for VTG that codes for the protein. Two favored strategies for evaluating fish VTG mRNA have risen, the ribonuclease insurance test (RPA) the quantitative invert interpretation polymerase chain response (QRT-PCR) 6

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Methods for Measuring VTG acceptance in fishes-Mass Spectrometry Mass Spectrometry (MALDI-MS) when all is said in done, MS ways to deal with protein measurement endeavor to gauge the protein to a great extent in its in place shape or depend on absorption systems (synthetic or enzymatic) to decrease the span of the protein into littler parts. The MS procedure permits both the immediate estimation of the VTG mass and era of peptide-fingerprinting information for further recognizable proof (Wunschel and Wahl, 2002). 7

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Participants in the Fathead Minnow VTG Study WA 2-19 Fathead Minnow members University of Florida, USA University of Idaho, USA Oregon State University, USA US EPA Duluth, USA University of Exeter, USA Brixham Environmental Laboratory, UK Battelle Richland, USA Battelle Sequim, USA Molecular Light Technology, UK Biosense, Norway INERIS, France Cemagref, France University of Windsor, Canada University of Southern Denmark, Denmark Finnish Environmental Institute, Finland 8

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Participants Zebrafish/Medaka VTG Study (contd) WA 2-26 Zebrafish/Medaka Participants Biosense Laboratories, Norway Center for Fish and Wildlife Health, University of Bern, Switzerland Department of Pathology, Vet. Drug, Swedish University of Agricultural Sciences, Sweden EnBioTec Laboratories, Ltd., Japan Environmental and Symbiotic Sciences, Prefectural University of Kumamoto, Japan Institute of Biology, University of Southern Denmark, Denmark Los Angeles County Sanitation District, USA National Institute for Environmental Studies (NIES), Japan Notox Safety & Environmental Research, the Netherlands Phylonix Pharmaceuticals, Inc., USA Unité d'Evaluation des Risques Ecotoxicologiques (INERIS), France. 9

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Objectives Fathead Minnow VTG Study Prepare standard arrangement of plasma, liver and entire body homogenates of fathead minnow ( Pimephales promelas ) to give a scope of VTG and mRNA fixations delivered in male and female fish ( in addition to a positive control) for assessment by partaking labs Determine the similarity of different strategies for the investigation of vitellogenin in fathead minnows by method for factual examination of the outcomes from eleven labs The outcomes from 8 ELISA research facilities, 3 mRNA labs and one Mass spectrometric (MS) strategy are displayed 10

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Objectives Zebrafish/Medaka VTG Study Prepare standard arrangement of liver and entire body homogenates of medaka and zebrafish for assessment by taking an interest labs; the arrangement speaks to a scope of vitellogenin focuses in male and female fish, in addition to a positive control Determine the equivalence of ELISA techniques for the investigation of vitellogenin of the two species by method for measurable examination of the outcomes from eleven labs Photo: Borg 2003 11

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Methods Fathead Minnow VTG Study (contd) Sample Preparation (Battelle) Obtained around 600 Fathead minnows Exposed subset of 100M and 190F to 300 ng/L 17 β estradiol in a 7-day static recharging treatment to initiate vitellogenin generation. Remaining fish (210M and 190F remained unexposed). Day 2 of introduction fish were yielded and liver tissue reaped for mRNA standard arrangement (80 EM/80 UM and 160EF/160UF) Prepared vitellogenin standard arrangement from plasma gathered from caudal vessels into heparinized hematocrit tubes. entire assemblage of uncovered and unexposed, male and female fish by crushing tissue with super cold ELISA examine support (1:1 proportion, fish:buffer by wt), centrifuging, speedy solidifying supernatant on fluid nitrogen Spiked unexposed male tissue of every species with known amount of decontaminated vitellogenin from comparing species as positive control 12

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Methods Zebrafish/Medaka VTG Study (contd) Sample Preparation Obtained around 400 zebrafish and 400 medaka Exposed subset of each gathering to 300 ng/L 17 β estradiol in a 7-day static recharging treatment to actuate vitellogenin generation Prepared vitellogenin standard arrangement from liver and entire assortment of uncovered and unexposed, male and female fish of every species by granulating tissue with super cold ELISA measure cushion (1:2 proportion, fish:buffer by wt), centrifuging, fast solidifying supernatant on fluid nitrogen Spiked unexposed male tissue of every species with known amount of cleansed vitellogenin from relating species as positive control ________________________ 1 Blood plasma was additionally gathered, however its examination was in this manner erased from study. 13

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Zebra angle Zebra angle S2 Zebra angle W2 Zebra angle L2 Methods Fathead, Zebrafish/Medaka VTG Study (contd) Shipment to taking an interest labs Subsampled 20-µL aliquots of homogenates and collected triplicate vials of each specimen sort as standard arrangement, every vial named with novel code for visually impaired examination: Shipped solidified at - 80°C in super-protected bundling to taking an interest labs on 2 June 2003 with documentation, data, and institutionalized chain-of-authority and information detailing frames Standard Series: Whole Body, Liver (W,L) uninduced male uninduced female instigated male actuated female positive control Purified vitellogenin as alignment standard for the species (S) 14

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Methods zebrafish Medaka VTG Study (contd) Sample investigation (taking an interest labs) zebrafish Sandwich catalyst immunoassay, VTG zebrafish counter acting agent (Biosense 2002) (6 labs) Sandwich ELISA, VTG zebrafish monoclonal immunizer (EnBio 2002; Nishi 2002) (1 lab) Direct noncompetitive sandwich ELISA, hostile to zebrafish lipovitellin, polyclonal neutralizer (Holbech et al. 2001) (1 lab) Modified Holbech et al. 2001 (Borg 2003, unpublished) (1 lab) [Competitive restricting test (Brion et al. 2002) (1 lab); information got past the point of no return for incorporation in measurable examination; see appendix] 15

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Methods Zebrafish/Medaka VTG Study (contd) Sample investigation (partaking labs) Medaka Sandwich catalyst immunoassay, VTG medaka neutralizer (Biosense 2003) (5 labs) Sandwich ELISA, VTG medaka monoclonal counter acting agent (EnBio 2002; Nishi 2002) (2 labs) Direct sandwich ELISA, VTG medaka monoclonal and biotinylated polyclonal antibodies (Transgenic 2002) (1 lab) [Competitive restricting test (Brion et al. 2002) (1 lab); information got past the point of no return for consideration in factual examination; see appendix] 16

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Methods Fathead Minnow VTG Study (contd) Sample investigation (taking an interest labs) Fathead Minnow ELISA (8 techniques, 11 taking an interest labs) Carp based polyclonal and monoclonal antibodies, sandwich ELISA (Biosense 2002) (3 labs) Carp based polyclonal antibodies, aggressive ELISA (1 lab) Carp based polyclonal and monoclonal antibodies, sandwich ELISA (1 lab) Trout based polyclonal antibodies in a focused ELISA (2 labs) Fathead minnow based polyclonal antibodies, focused, immune response catch (1 lab) Fathead minnow based, monoclonal counter acting agent, coordinate ELISA (1 lab) Zebrafish based polyclonal antibodies, aggressive ELISA (1 lab) Zebrafish based hostile to lipovitellin coordinate non-focused sandwich ELISA (1 lab) mRNA (3 strategies, 3 taking an interest labs) mRNA - RT-PCR mRNA - qRT-PCR TaqMan mRNA - HPA (hybridization assurance test) Mass Spectrometric (1 strategy, 1 taking part lab) Matrix helped laser desorption/ionization mass spectrometry (MALDI-MS) (1 lab) 17

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Results Zebrafish/Medaka VTG Study 1 uninduced male < 2 uninduced female < 3 actuated male < 4 instigated female >> 5 positive control Expected pattern was as per the following: Results indicated great relative following of pattern, especially in medaka, however higher inconstancy in total measured values Best fit: medaka liver outcomes A: zebrafish liver B: zebrafish entire body Code 0 = Blank; Code 1 = Uninduced Male; Code 2 = Uninduced Female; Code 3 = Induced male; Code 4 = Induced Female; Code 5 = Positive Control C: Medaka liver D. Medaka wh

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