Comment[ArrayExpressAccession]	E-GEOD-7837
Public Release Date	2007-08-31
Investigation Title	Mechanism for hepatic tumor promotion by PFOA in rainbow trout																																										
Comment[Submitted Name]	Mechanism for hepatic tumor promotion by PFOA in rainbow trout
Experiment Description	Perfluorooctanoic acid (PFOA) is a potent hepatocarcinogen and peroxisome proliferator (PP) in rodents.  Humans are not susceptible to peroxisome proliferation and are thought to be refractory to carcinogenesis by PFOA and other PPs.  However, previous studies with rainbow trout have shown that they are also insensitive to peroxisome proliferation by the PP, dehydroepiandrosterone (DHEA), but are still susceptible to enhanced hepatocarcinogenesis after chronic exposure.  In this study, we determined whether PFOA is also a tumor promotor in trout and then examined hepatic gene expression profiles to further investigate possible mechanisms of action.  Trout were initiated as fry to the hepatocarcinogen, aflatoxin B1, and then fed 200-1800 ppm PFOA in the diet for 30 weeks.  Two structurally diverse PPs, clofibrate (CLOF) and DHEA, were included for comparison.  Hepatic gene expression profiles were subsequently examined in animals exposed to similar doses of PFOA, DHEA and CLOF along with 5 ppm 17β-estradiol (E2; a known tumor promotor) in the diet.  PFOA (1800 ppm) and DHEA treatments resulted in enhanced liver tumor incidence and multiplicity while CLOF showed no effect.  Carcinogenesis seemed independent of peroxisome proliferation as no induction of peroxisomal β-oxidation and catalase activity were observed.  Alternately, plasma VTG was elevated in fish fed PFOA and DHEA suggesting that estrogenic mechanisms may play a role.  Both tumor promotors, PFOA and DHEA, resulted in strong correlation of transcriptional profiles with E2 by Pearson correlation (R=0.81 and 0.78, respectively).  In comparison, CLOF regulated no genes in common with E2.  Overall, these data suggest that the tumor promoting activities of DHEA and PFOA in trout are independent of peroxisome proliferation and may involve estrogenic mechanisms. Juvenile trout, 12-18 months old, were fed experimental diets containing 500 or 1800 ppm PFOA, 1800 ppm CLOF, 750 ppm DHEA, 5 ppm E2 or 0.15 % dimethyl sulfoxide vehicle control for 14 days.  Liver samples were collected for microarray analysis.  Hybridizations were performed using standard reference design with dye-swapping. For each sample, equal amounts of RNA (µg) were pooled from five fish per tank for every treatment (n=3 biological replicates per treatment). cDNA from two of the three biological replicates was dye-swapped and hybridized to two slides as technical replicates (5 arrays per treatment).																																										
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Term Source Name	EFO
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Term Source File	http://efo.svn.sourceforge.net/viewvc/efo/trunk/src/efoinowl/efo.owl																																										
Person Last Name	Tilton	Tilton	Orner	Benninghoff	Hendricks	Pereira	Williams																																				
Person First Name	Susan	Susan	Gayle	Abby	Jerry	Cliff	David																																				
Person Mid Initials	C.	C	A	D	D	B	E																																				
Person Email	tiltons@u.washington.edu																																										
Person Affiliation	Oregon State University																																										
Person Phone	541-737-6498																																										
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Person Address	Environmental and Molecular Toxicology, Oregon State University, 1007 ALS Bldg., Corvallis, OR, USA																																										
Person Roles	submitter																																										
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Protocol Name	P-GSE7837-1	P-GSE7837-8	P-GSE7837-27	P-GSE7837-40	P-GSE7837-36	P-GSE7837-31	P-GSE7837-29	P-GSE7837-7	P-GSE7837-34	P-GSE7837-16	P-GSE7837-41	P-GSE7837-11	P-GSE7837-39	P-GSE7837-10	P-GSE7837-23	P-GSE7837-24	P-GSE7837-37	P-GSE7837-18	P-GSE7837-28	P-GSE7837-15	P-GSE7837-21	P-GSE7837-33	P-GSE7837-22	P-GSE7837-35	P-GSE7837-25	P-GSE7837-42	P-GSE7837-12	P-GSE7837-43	P-GSE7837-6	P-GSE7837-19	P-GSE7837-13	P-GSE7837-17	P-GSE7837-30	P-GSE7837-38	P-GSE7837-20	P-GSE7837-14	P-GSE7837-2	P-GSE7837-26	P-GSE7837-5	P-GSE7837-32	P-GSE7837-3	P-GSE7837-4	P-GSE7837-9
Protocol Description	ID_REF = <br>ch1 Area = Number of pixels in the local signal region<br>ch2 Area = Number of pixels in the local signal region<br>VALUE = Log2 Ratio [(ch1 Mean - ch1 Background)/(ch2 Mean - ch2 Background)] of raw data<br>ch1 Signal Noise Ratio = Signal Noise Ratio<br>ch2 Signal Noise Ratio = Signal Noise Ratio<br>ch1 Mean = Mean pixel intensity computed over the local signal region<br>ch1 Background = Mean pixel intensity computed over the local background region<br>ch2 Mean = Mean pixel intensity computed over the local signal region<br>ch2 Background = Mean pixel intensity computed over the local background region<br>ch1 Mean Std Dev = Standard deviation of pixel intensities over the local signal region<br>ch1 Background Std Dev = Standard deviation of pixel intensities over the local background region<br>ch2 Mean Std Dev = Standard deviation of pixel intensities over the local signal region<br>ch2 Background Std Dev = Standard deviation of pixel intensities over the local background region	Scanned images (5 um) were acquired with ScanArray Express (PerkinElmer, Boston, MA) at an excitation of 543 nm for Cy3 and 633 nm for Cy5 and at 90% power. The photomultiplier tube (PMT) settings for each fluor were set based on intensity of spiked internal alien controls to normalize among all slides in the experiment. Image files were quantified in QuantArray (PerkinElmer) and raw median signal and background values were exported to BioArray Software Environment (BASE; Saal et al. 2002) for analysis. 	Slide# Omy005.P94.13166413	Slide# Omy005.P94.13171504	Slide# Omy005.P94.13171129	Slide# Omy005.P94.13171126	Slide# Omy005.P94.13171142	Slide# Omy005.P94.13166409	Slide# Omy005.P94.13171500	Slide# Omy005.P94.13171486	Slide# Omy005.P94.13171132	Slide# Omy005.P94.13171492	Slide# Omy005.P94.13171503	Slide# Omy005.P94.13166411	Slide# Omy005.P94.13171487	Slide# Omy005.P94.13171488	Slide# Omy005.P94.13171141	Slide# Omy005.P94.13171498	Slide# Omy005.P94.13171485	Slide# Omy005.P94.13171484	Slide# Omy005.P94.13166410	Slide# Omy005.P94.13171501	Slide# Omy005.P94.13166412	Slide# Omy005.P94.13171128	Slide# Omy005.P94.13166626	Slide# Omy005.P94.13171134	Slide# Omy005.P94.13171494	Slide# Omy005.P94.13171137	Hybridizations were performed with the Genisphere Array 350 kit and instructions (Hatfield, PA) using standard reference design with dye-swapping. Briefly, 7 ug total RNA were reverse-transcribed with Superscript II (Invitrogen) using the Genisphere oligo d(T) primer containing a capture sequence for the Cy3 or Cy5 labelling reagents. Each reaction was spiked with a range of concentrations (0.0049 – 2.5 ng/ul) of the ten SpotReport Alien Oligo controls (Stratagene). Each cDNA sample containing the capture sequence for the Cy3 or Cy5 label was combined with equal amounts of reference cDNA (pooled from sham-initiated controls) containing the capture sequence for the opposite label. cDNA from two of the three biological replicates were dye-swapped and hybridized to two slides as technical replicates. cDNA from the reference sample was also hybridized to dye-swapped slides (against itself) following the same protocol as experimental samples for use as a negative control. Prior to hybridization, microarrays were processed post-printing by washing twice in 0.1% SDS for 5 min, 2X SSC, 0.1% SDS at 47C for 20 min, 0.1X SSC for 5 min, water for 3 min, then dried by centrifugation. The cDNAs (25 ul) were hybridized to arrays in formamide buffer [50% formamide, 8X SSC, 1% SDS, 4X Denhardt’s solution] for 16 h at 47C with 22x25 mm Lifterslips (Erie Scientific, Portsmouth, NH). Arrays were then washed once in 2X SSC, 0.1% SDS at 47C for 10 min, twice in 2X SSC, 0.1% SDS for 5 min, twice in 1X SSC for 5 min, twice in 0.1X SSC for 5 min and dried by centrifugation. Shaded from light, the Cy3 and Cy5 fluorescent molecules (3DNA capture reagent, Genisphere) were hybridized in formamide buffer for 3 h at 49C to the corresponding capture sequences on cDNAs bound to the arrays. Arrays were washed in the dark with SSC containing 0.1 M DTT and dried as described earlier.	Slide# Omy005.P94.13171125	Slide# Omy005.P94.13171502	Slide# Omy005.P94.13171495	Slide# Omy005.P94.13171136	Juvenile trout, 12-18 months old, were maintained in separate 375-L tanks (n=3 tanks) for each treatment with 5 fish per tank.  Animals were fed a maintenance ration (2.8% w/w) of experimental diets containing 0.15%  DMSO for 14 days.  On day 15, fish were euthanized by deep anesthesia with 250 ppm tricaine methanesulfonate.  Approximately 100 mg liver tissue from individual fish was minced, stored in TRIzol Reagent (Invitrogen, Carlsbad, CA) and quick frozen in liquid nitrogen for gene expression analysis.	Juvenile trout, 12-18 months old, were maintained in separate 375-L tanks (n=3 tanks) for each treatment with 5 fish per tank.  Animals were fed a maintenance ration (2.8% w/w) of experimental diets containing 5 ppm E2 for 14 days.  On day 15, fish were euthanized by deep anesthesia with 250 ppm tricaine methanesulfonate.  Approximately 100 mg liver tissue from individual fish was minced, stored in TRIzol Reagent (Invitrogen, Carlsbad, CA) and quick frozen in liquid nitrogen for gene expression analysis.	Juvenile trout, 12-18 months old, were maintained in separate 375-L tanks (n=3 tanks) for each treatment with 5 fish per tank.  Animals were fed a maintenance ration (2.8% w/w) of experimental diets containing 1800 ppm PFOA for 14 days.  On day 15, fish were euthanized by deep anesthesia with 250 ppm tricaine methanesulfonate.  Approximately 100 mg liver tissue from individual fish was minced, stored in TRIzol Reagent (Invitrogen, Carlsbad, CA) and quick frozen in liquid nitrogen for gene expression analysis.	Juvenile trout, 12-18 months old, were maintained in separate 375-L tanks (n=3 tanks) for each treatment with 5 fish per tank.  Animals were fed a maintenance ration (2.8% w/w) of experimental diets containing 500 ppm PFOA for 14 days.  On day 15, fish were euthanized by deep anesthesia with 250 ppm tricaine methanesulfonate.  Approximately 100 mg liver tissue from individual fish was minced, stored in TRIzol Reagent (Invitrogen, Carlsbad, CA) and quick frozen in liquid nitrogen for gene expression analysis.	Juvenile trout, 12-18 months old, were maintained in separate 375-L tanks (n=3 tanks) for each treatment with 5 fish per tank.  Animals were fed a maintenance ration (2.8% w/w) of experimental diets containing 1800 ppm clofibrate for 14 days.  On day 15, fish were euthanized by deep anesthesia with 250 ppm tricaine methanesulfonate.  Approximately 100 mg liver tissue from individual fish was minced, stored in TRIzol Reagent (Invitrogen, Carlsbad, CA) and quick frozen in liquid nitrogen for gene expression analysis.	Juvenile trout, 12-18 months old, were maintained in separate 375-L tanks (n=3 tanks) for each treatment with 5 fish per tank.  Animals were fed a maintenance ration (2.8% w/w) of experimental diets containing 0.15% DMSO for 14 days.  On day 15, fish were euthanized by deep anesthesia with 250 ppm tricaine methanesulfonate.  Approximately 100 mg liver tissue from individual fish was minced, stored in TRIzol Reagent (Invitrogen, Carlsbad, CA) and quick frozen in liquid nitrogen for gene expression analysis.	Juvenile trout, 12-18 months old, were maintained in separate 375-L tanks (n=3 tanks) for each treatment with 5 fish per tank.  Animals were fed a maintenance ration (2.8% w/w) of experimental diets containing 750 ppm dehydroepiandrosterone for 14 days.  On day 15, fish were euthanized by deep anesthesia with 250 ppm tricaine methanesulfonate.  Approximately 100 mg liver tissue from individual fish was minced, stored in TRIzol Reagent (Invitrogen, Carlsbad, CA) and quick frozen in liquid nitrogen for gene expression analysis.	Invitrogen TRIzol extraction followed by cleanup with Qiagen RNeasy according to vendor protocol.	Labeled using Genisphere Array 350 kit adapted from manufacturer protocol.	Raw mean signal and background values are included. For experiment, data were background subtracted and normalized by LOWESS. Stringent criteria were used to filter for genes that were regulated at least 1.8-fold consistently in all features from biological replicates (n=3 per treatment) and had a p-value <0.05 by Welch’s t-test.
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Protocol Type	bioassay_data_transformation	image_aquisition	hybridization	hybridization	hybridization	hybridization	hybridization	hybridization	hybridization	hybridization	hybridization	hybridization	hybridization	hybridization	hybridization	hybridization	hybridization	hybridization	hybridization	hybridization	hybridization	hybridization	hybridization	hybridization	hybridization	hybridization	hybridization	hybridization	hybridization	hybridization	hybridization	hybridization	hybridization	specified_biomaterial_action	specified_biomaterial_action	specified_biomaterial_action	specified_biomaterial_action	specified_biomaterial_action	specified_biomaterial_action	specified_biomaterial_action	nucleic_acid_extraction	labeling	feature_extraction
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Experimental Factor Name	AGE	TREATMENT	STRAIN																																								
Experimental Factor Type	Age	Treatment	Strain																																								
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Comment[SecondaryAccession]	GSE7837																																										
Comment[GEOLastUpdateDate]	2007-05-17
Comment[GEOReleaseDate]	2007-08-30
Comment[ArrayExpressSubmissionDate]	2007-05-16
SDRF File	E-GEOD-7837.sdrf.txt