Genomic, proteomic, and metabolomic technologies continue to receive increasing interest from environmental toxicologists.  This interest is due to the great potential of these technologies to identify detailed modes of action and to provide assistance in the evaluation of a contaminantÂs risk to aquatic organisms.  Our experimental model is the zebrafish (Danio rerio) exposed to reference endocrine disrupting compounds in order to investigate compound-induced changes in gene transcript profiles.  Adult, female zebrafish were exposed to 0, 15, 40, and 100 ng/L of 17 alpha-ethynylestradiol (EE2) and concentration and time-dependent changes in hepatic gene expression were examined using Affymetrix GeneChipÂ® Zebrafish Genome Microarrays.   At 24, 48, and 168 hours, fish were sacrificed and liver mRNA was extracted for gene expression analysis (24 and 168 hours only).  In an effort to link gene expression changes to effects on higher levels of biological organization, body and ovary weights were measured and blood was collected for measurement of plasma steroid hormones (17 beta-estradiol (E2), testosterone (T)) and vitellogenin (VTG) using ELISA.   EE2 exposure significantly affected GSI, E2, T, VTG and gene expression.  We observed 1575 genes that were significantly affected (up- or down-regulated by at least 1.5-fold (p ? 0.001) in a concentration-dependent manner by EE2 exposure at either 24 or 168 hours.  EE2 exposure altered transcription of genes involved in steroid hormone homeostasis, cholesterol homeostasis, retinoic acid metabolism, and cell growth and proliferation.  Plasma VTG was significantly increased at 24, 48, and 168 hours (p<0.05) at 40 and 100 ng/L and at 15 ng/L at 168 hours.  E2 and T were significantly reduced following EE2 exposure at 48 and 168 hours.  GSI was decreased in a dose-dependent manner at 168 hours.  In this study, we identified genes involved in a variety of biological functions that have the potential to be used as markers of exposure to estrogenic substances.  Future work will evaluate the use of these genes in zebrafish exposed to weak estrogens to determine if these genes are indicative of exposure to estrogens with varying potencies.

Transcription profiling of zebrafish livers following exposure to 17 alpha-ethynylestradiol (EE2) at 0, 15, 40, or 100 ng/L

Hepatic gene expression profiling using Genechips in zebrafish exposed to 17&#945;-ethynylestradiol

Affymetrix GeneChip Zebrafish Genome Array [Zebrafish]

Hepatic gene expression profiling using Genechips in zebrafish exposed to 17α-ethynylestradiol

rawData

scan

processedDataMatrix

Genomic, proteomic, and metabolomic technologies continue to receive increasing interest from environmental toxicologists.  This interest is due to the great potential of these technologies to identify detailed modes of action and to provide assistance in the evaluation of a contaminant’s risk to aquatic organisms.  Our experimental model is the zebrafish (Danio rerio) exposed to reference endocrine disrupting compounds in order to investigate compound-induced changes in gene transcript profiles.  Adult, female zebrafish were exposed to 0, 15, 40, and 100 ng/L of 17 alpha-ethynylestradiol (EE2) and concentration and time-dependent changes in hepatic gene expression were examined using Affymetrix GeneChip® Zebrafish Genome Microarrays.   At 24, 48, and 168 hours, fish were sacrificed and liver mRNA was extracted for gene expression analysis (24 and 168 hours only).  In an effort to link gene expression changes to effects on higher levels of biological organization, body and ovary weights were measured and blood was collected for measurement of plasma steroid hormones (17 beta-estradiol (E2), testosterone (T)) and vitellogenin (VTG) using ELISA.   EE2 exposure significantly affected GSI, E2, T, VTG and gene expression.  We observed 1575 genes that were significantly affected (up- or down-regulated by at least 1.5-fold (p ? 0.001) in a concentration-dependent manner by EE2 exposure at either 24 or 168 hours.  EE2 exposure altered transcription of genes involved in steroid hormone homeostasis, cholesterol homeostasis, retinoic acid metabolism, and cell growth and proliferation.  Plasma VTG was significantly increased at 24, 48, and 168 hours (p<0.05) at 40 and 100 ng/L and at 15 ng/L at 168 hours.  E2 and T were significantly reduced following EE2 exposure at 48 and 168 hours.  GSI was decreased in a dose-dependent manner at 168 hours.  In this study, we identified genes involved in a variety of biological functions that have the potential to be used as markers of exposure to estrogenic substances.  Future work will evaluate the use of these genes in zebrafish exposed to weak estrogens to determine if these genes are indicative of exposure to estrogens with varying potencies.

compound

dose

time

Transcription profiling by array of zebrafish liver after treatment with 17 alpha-ethynylestradiol

Genomic, proteomic, and metabolomic technologies continue to receive increasing interest from environmental toxicologists.  This interest is due to the great potential of these technologies to identify detailed modes of action and to provide assistance in the evaluation of a contaminant’s risk to aquatic organisms.  Our experimental model is the zebrafish (Danio rerio) exposed to reference endocrine disrupting compounds in order to investigate compound-induced changes in gene transcript profiles.  Adult, female zebrafish were exposed to 0, 15, 40, and 100 ng/L of 17 alpha-ethynylestradiol (EE2) and concentration and time-dependent changes in hepatic gene expression were examined using Affymetrix GeneChip® Zebrafish Genome Microarrays.   At 24, 48, and 168 hours, fish were sacrificed and liver mRNA was extracted for gene expression analysis (24 and 168 hours only).  In an effort to link gene expression changes to effects on higher levels of biological organization, body and ovary weights were measured and blood was collected for measurement of plasma steroid hormones (17 beta-estradiol (E2), testosterone (T)) and vitellogenin (VTG) using ELISA.   EE2 exposure significantly affected GSI, E2, T, VTG and gene expression.  We observed 1575 genes that were significantly affected (up- or down-regulated by at least 1.5-fold (p ? 0.001) in a concentration-dependent manner by EE2 exposure at either 24 or 168 hours.  EE2 exposure altered transcription of genes involved in steroid hormone homeostasis, cholesterol homeostasis, retinoic acid metabolism, and cell growth and proliferation.  Plasma VTG was significantly increased at 24, 48, and 168 hours (p<0.05) at 40 and 100 ng/L and at 15 ng/L at 168 hours.  E2 and T were significantly reduced following EE2 exposure at 48 and 168 hours.  GSI was decreased in a dose-dependent manner at 168 hours.  In this study, we identified genes involved in a variety of biological functions that have the potential to be used as markers of exposure to estrogenic substances.  Future work will evaluate the use of these genes in zebrafish exposed to weak estrogens to determine if these genes are indicative of exposure to estrogens with varying potencies.


Affymetrix:Protocol:ExpressionStat

Title: Affymetrix CHP Analysis (ExpressionStat). Description:

Affymetrix:Protocol:Hybridization-EukGE-WS2v4_450

Title: Affymetrix EukGE-WS2v4_450 Hybridization. Description:

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-TABM-472

Total RNA Isolation from Tissues Affymetrix GeneChip Rev 3.4.2 Trizol Reagent RNA Isolation: 1) Place tissue (up to 100 mg) in 1 ml of Trizol and homogenize using Polytron with 12mm generator for 30 seconds, 30,000 rpm in a Falcon 15ml polypropylene tube. *Keep samples on ice after homogenization (Optional depending on tissue type: May also use Beadmill for no more then 10min on 30 mH, outside row, 5mm stainless steal bead (Qiagen), in a 2ml USA Scientific, Inc. tube. Cat#1620-2700) 2) Incubate 5 min at room temp to permit complete dissociation of nucleoprotein complexes. 3) Centrifuge at 12,000xg for 10 minutes at 4„aC in order to remove insolubles. 4) Remove supernatant to new 1.5ml tube. 5) Add 0.2 ml chloroform to 1 ml lysate (depending on tissue mass more chloroform may be added - facilitates phase separation on PLG tubes) Shake vigorously for 15 seconds. 6) Incubate 2-3 min at room temp. 7) Transfer to 2.0 ml heavy phase lock gels(brinckman/eppendorf) and spin in microfuge 12,000-16,000 x g for 5 min at 4 degrees C. (note:spin down PLG tube for 20-30sec before adding sample and do not vortex after sample has been added onto PLG tube) 8) Remove upper aqueous phase to fresh tube. Volume of aqueous phase will be ~600-700 ul. 9) Add 600-700 ul (equal volume) of 100% isopropanol to samples, vortex and incubate 10 min at room temp. 10) Spin in microfuge at 16,000 x g for 10 min at 4 degrees C (Only do steps 11&17 if samples were stored in RNA later and there is a biphase pellet) 11) Aspirate/decant isopropanol from pelleted material and wash pellet with 500 ul 50% isopropanol (important for samples stored in RNAlater solution) 12) Spin in microfuge at 16,000 x g for 10 min at 4„aC 13) Aspirate/decant isopropanol and overlay pellets with 1 ml -20 degrees C 70% EtOH. 14) Spin in microfuge at 16,000 x g for 5 min at 4 degrees C. 15) Aspirate/decant EtOH and air dry pellets. Do not Speed-Vac. 16) Resuspend pellets in 100-200 ul DEPC-H20. Make sure pellets are completely in solution before storage. 17) Heat samples following step 16 for 5 min at 65 degrees C, if they are not going into solution. This is to ensure the pellets are in solution. 18) Determine A260 and A260/A280 of unclean RNA. This is important to verify complete resuspension of RNA pellets and to avoid overloading column during RNesy Cleanup (no more then 100ug). RNeasy Mini Cleanup for RNA Cleanup Note: RNA is cleaned up using QIAgen¡¦s RNeasy Cleanup protocol. To avoid overloading RNeasy columns do not clean more than 100 ug of RNA per column. All spins done at room temperature. 1) Before use, mix 10 ull of ƒÒ-mercaptoethanol with 1 ml of RLT Buffer. Make sufficient amount of RLT+Beta-ME for isolations. 2) Mix 100 ul isolated RNA (we prefer a total of 70 ug) with 350 ul RLT+Beta-ME. 3) Add 250 ul of 100% EtOH and vortex. 4) Apply sample (700 ul) to RNeasy spin column and spin 15 sec in microfuge at max for rotor. 5) Reapply flow-through to column and spin again for 15 sec in microfuge at max for rotor. 6) Transfer column to new 2 ml collection tube and discard flow-through. 7) Add 500 ul RPE Buffer to column and spin 15 sec in microfuge at max for rotor. 8) Discard flow-through. 9) Add 500 ul more RPE to column and spin 2 minutes in microfuge at max for rotor. It is important to make sure there is no residual RPE left on the column membrane, since this can decrease yield of purified RNA recovered during elution. 10) Transfer column to fresh 1.5 ml collection tube and add 30-40 ul RNase-free waterheated to 95 degrees C (provided in kit). (Optional: if yields are low, another elution of 30-40ul RNase-free water over the column should be done. The second elution should be quantified (A260 ) separately from the first.) 11) Incubate 5 min at room temp. 12) Spin 1 min in microfuge to collect eluant. 13) Determine A260 and A260/A280 of RNA Also run an Agilent chip using 250ng to verify integrity of ribosomal bands and quantitation. (Follow standard total RNA Nano Assay protocol from Agilent) Supplies: RNAlater (Ambion cat #7020) Trizol (Invitrogen cat #15596) Chloroform (Sigma cat #C-2432) Phase Lock Gel Tubes, Heavy 2.0 ml (Eppendorf cat #0032 005.152) Isoporapanol (EM science cat #PX-1835) 70% ethanol DEPC water (Ambion cat #9915G) Nuclease-free water (Ambion cat #9037) RNeasy Mini Kit (Qiagen cat #74106) £]-mercaptoethanol (Sigma cat #M3148) Agilent Nano Assay cat#5065-4476

P-TABM-473

Adult male and female zebrafish (>121 days) were obtained from Aquatic Research Organisms (Hampton, NH) and maintained together (1 male: 2 female) for at least 2 weeks prior to experimentation, according to Procter and Gamble’s animal care-approved protocols. Fish were kept together in 75 L tanks that received a constant flow of well water under a 16:8 hour, Light:Dark photoperiod. Temperature and dissolved oxygen were measured daily in each aquaria during the experiment while pH, hardness, conductivity, alkalinity, and ammonia were measured on the first and last day of the experiment. Fish were fed a combination of newly hatched brine shrimp and Tetramin® (Tetra, Germany) flakes twice a day, ad libitum.

P-TABM-474

Target Synthesis Protocol for Affymetrix GeneChip *For High Throughput processing (*HT) use Matrix Impact2 8 channel expandable pipettes for liquid handling *Progam themal cycler for each individual synthesis step. I. First Strand cDNA Synthesis: 1. In an RNAse-free 1.5 ml Eppendorf tube make master mix of primer & spike to add to 10ug of clean total RNA. (Do not use 8- channel pipet for *HT processing of first strand master mixes) Use MJ research 8 strip or 96 well plates to perform the reaction. 1 ul (100 pmol) T7-(dT)24 primer 2 ul Synthesis Spike (depends on input RNA read affy Poly A ctrl kit protocol) 2. Add 3ul Master mix to 8ul (10ug) clean total RNA. Final volume 11ul 3. Incubate for 10 min at 70 degrees C in thermal cycler. 4. Quick spin and place on ice for 7-10min. 5. Add: Using a Master Mix 4 ul 5X First Strand Buffer 2 ul 0.1 M DTT 1 ul 10 mM dNTP Mix 2 ul SuperScript II Reverse Transcriptase (total volume = 20 ul). 6. Incubate for 1 hr at 42 degrees C in thermal cycler. II. Second Strand DNA Synthesis: 1. Place first strand reaction tubes on ice 10 min and quick spin. 2. Add: Using a Master Mix 91 ul DEPC-H20 30 ul 5X Second Strand Buffer 3 ul 10 mM dNTP Mix 1 ul 10 U/ul E. coli DNA Ligase 4 ul 10 U/ul E. coli DNA Polymerase I 1 ul 2 U/ul RNAse H 3. Add 130ul Master Mix(*HT). Mix by pipetting and quick spin. 4. Incubate for 2 hr at 16degrees C in thermal cycler. 5. Store at -20 degrees C or proceed to cDNA Cleanup. If continuing keep rxn on ice for at least 10 min. III. cDNA Cleanup: 1. Pellet Phase Lock Gel (PLG-Light) by spinning in microfuge for 20-30 seconds. 2. To cDNA samples, add an equal volume (162 ul) of (25:24:1) phenol:chloroform:isoamyl alcohol and vortex.(perform individually) 3. Transfer sample to pelleted PLG tubes (do not vortex). 4. Spin 2 min in microfuge. 5. Transfer upper (aqueous) phase to fresh 1.5 ml Eppendorf tubes one by one. 6. Add 1 ul of NF PelletPaint, 0.5 vol (80 ul) of 7.5 M Ammonium Acetate, and 2.5 vol (400 ul) -20 degrees C 100% EtOH. (For *HT make master mix then add) 7. Immediately spin in microfuge for 20 min at room temp. 8. Aspirate/Decant EtOH and overlay pellet with 0.5 ml –20 degrees C 70% EtOH.(*HT) 9. Spin 2 min at room temp. 10. Aspirate/Decant EtOH and air-dry pellet (a Speed-Vac may be used to accelerate drying). 11. Resuspend pellet in 10.5 ul DEPC-H2O(*HT). Important: After adding 10.5ul of DEPC water, place tubes on ice for 10 min. To ensure that cDNA is completely resuspended, flick tubes,spin down & heat 5min at 65 degrees C, Do not mix by pipetting. This procedure is done to ensure complete resuspension of cDNA. This step is critical for high yields of cRNA. 12. Remove 0.5 ul for gel analysis and store remainder at -20 degrees C or proceed to cRNA Synthesis. IV. Gel Analysis of cDNA: 1. Use 0.5 ul of each cDNA aliquot for analysis using the Agilent 2100 Bioanalyzer Total RNA Nano assay. It is not necessary to quantitate the cDNA by A260. cDNA samples should look identical on gel, which indicates proper resuspension/relative yields of cDNA. Ribosomal RNA bands will, and should be present in cDNA samples run on the gel. 2. If cDNA is of sufficient quality, proceed to cRNA Synthesis. V. cRNA Synthesis: 1. Using the Enzo BioArray High Yield RNA Transcription Labeling Kit mix the following at room temp in the listed order: (Ensure extra RXNs in master mix for *HT addition) All reagents, except DTT, should be kept on ice before adding. 10.0ul cDNA Add:Using Master Mix 30 ul (preform at room temp) 12ul DEPC Water 4ul 10x Hy Rxn Buffer 4ul 10x Biotin Labeled Ribonucleotides 4ul 10x DTT (bring to room temp to prevent precipitation) 4ul 10x RNase Inhibitor Mix 2ul 20x T7 RNA Polymerase 40ul Total Volume 2. Incubate 6 hr at 37 degrees C in thermal cycler. 3. Place tubes on ice (10min) or at -20 degrees C for long term storage, following the incubation VI. cRNA Cleanup: (see detailed protocol for HT cleanup) Note: RNA is cleaned up using Qiagen’s RNeasy mini cleanup protocol 1. In a RNAse-free 1.5 ml Eppendorf tube, mix 40 ul of cRNA(from step V) with 60 ul DEPC-H2O. 2. For 4 purifications, mix 2 ml of RLT buffer with 20 ul Beta mercaptoethanol to make RLT+Beta-ME stock. 3. Add 350 ul RLT+Beta ME stock to each cRNA tube. 4. Add 250 ul 100% EtOH and vortex. (Note do not spin down!) 5. Apply sample to RNeasy column. 6. Spin 15 sec in microfuge. 7. Reapply flow-through to column and spin another 15 sec in microfuge. 8. Transfer column to fresh collection tube and dispose of flow-through. 9. Add 500 ul RPE buffer to column and spin 15 sec in microfuge. 10. Discard flow-through. 11. Add another 500 ul RPE buffer to column and spin 2 min in microfuge. It is important to make sure there is no residual RPE left on the column membrane, since this can decrease yield of purified RNA recovered during elution. 12. Transfer column to fresh 1.5 ml collection tube. 13. Add 30 -40ul RNAseFree water heated to 95 degrees C to column (make sure water is dispensed onto filter within the column), and incubate 5 min at room temp. (Optional: if yields are low, another elution of 30-40 ul RNase-free water over the column should be done. The second elution should be quantified (A260) separately from the first). 14. Spin 1 min to elute cRNA. 15. Determine A260 and A260/A280 of RNA. Also run an Agilent chip using 250ng to verify integrity of ribosomal bands and quantification. (Follow standard total RNA Nano Assay protocol from Agilent)A260/A280 ratio (should be 1.9-2.0). 16. If cRNA is not at least 0.6 ug/ul, concentrate sample in Speed-Vac. VII. cRNA Fragmentation: 1. Use MJ research 8 strip or 96 well plates to perform the reaction X ul (20 ug) cRNA 8 ul 5X Fragmentation Buffer (*HT) X ul DEPC-H2O 40 ul TOTAL 2. Incubate 94 degrees C for 35 min. 3. Place on ice. 4. Remove 1 ul of fragmented cRNA for gel analysis. 5. Store remainder of fragmented cRNA at -20 degrees C. Fragmented cRNA may be stored at -20 degrees C for months before hybridization to GeneChip. VIII. Gel Analysis of cRNA: 1. Using the Agilent 2100 Bioanlayzer Total RNA Nano Assay use 250ng aliquot of unfragmented cRNA from section VI, step 15 and a 1 ul aliquot of fragmented cRNA from section VII, step 4. 2. If cRNA is of sufficient quality, proceed to target hybridization.

P-TABM-475

The Affymetrix Microarray Suite software (Version 1.3) uses probe pair signal intensities to make present, absent, and marginal calls for each gene based on detection algorithms outlined in the GeneChip® Expression Analysis Overview Manual (Affymetrix). Genes that were identified as absent in all samples across treatment groups were eliminated from further comparative analysis. Expression measures were analyzed in S-plus software (Insightful, Seattle WA). The effects of dose and time were investigated using various linear models (Venables and Ripley, 2006) and nonparametric analyses (Gibbons and Chakraborti, 2003). Linear models were fit that accounted for the effects of dose, duration of follow-up, and their interactions. In these models, dose was treated as both a nominal factor, and as a regressor for the purpose of detecting dose-response. In the nonparametric analyses, trends in gene expression response to dose and time were evaluated by using a stratified test for trend. Stratification is used to control for effects of nuisance variables on responses. Pairwise comparisons of treated groups to their time-matched controls were also used to further understand the dominant patterns of dose effects. Differences in GSI, Vtg, T, and E2 among treatment groups were identified using one-way analysis of variance (ANOVA) followed by Tukey’s honestly significant difference test (JMP IN version 5.1.2, SAS Institute, USA). Fold changes were determined for each gene by calculating the ratio of the mean expression values of the control and treated samples.

P-TABM-476

A stock solution of 5.88 mg/L EE2 (17 alpha-Ethynylestradiol (CAS #:57-63-6; Purity ? 98% high-performance liquid chromatography; Sigma Aldrich, St. Louis, MO) was prepared in ethanol (Aaper Alcohol and Chemical Co., Shelbyville, KY). Aquaria delivery solutions were prepared by diluting the stock solution in deionized water with ethanol as the solvent carrier To synchronize the reproductive status of female fish, males and females were separated five days prior to the initiation of exposure. Gravid females were exposed to EE2 at nominal concentrations of 0, 15, 40, or 100 ng/L under flow-through conditions in 33 L aquaria for 24, 48 or 168 hours. Each aquaria contained 15 female zebrafish. Flow rate of dilution water to the exposure aquaria was maintained at 100 ml/minute using flow controllers (Alicat Scientific, Tucson, AZ) yielding 4.4 aquaria renewals per day. To achieve the desired concentrations, EE2 and the solvent control (ethanol) were delivered to the exposure aquaria using programmable glass syringe pumps (Harvard Apparatus PHD2000, Holliston, MA) at a flow-rate of 40 ml/hour. Prior to delivery to the exposure aquaria, EE2 and the solvent control were combined with the dilution water at a “T” fitting. During the exposure, fish were fed twice daily, once with newly hatched brine shrimp and once Tetramin® (Tetra, Germany) to reduce the effect of a change in diet on gene expression. Debris was siphoned from each tank daily.

Transcription profiling of zebrafish livers following exposure to 17 alpha-ethynylestradiol (EE2) at 0, 15, 40, or 100 ng/L

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