Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcript profiles of PFAAs in trout liver

ABSTRACT: Background: Previously, we reported that perfluorooctanoic acid (PFOA) promotes liver cancer in manner similar to that of 17β-estradiol (E2) in rainbow trout. Also, other perfluoroalkyl acids (PFAAs) are weakly estrogenic in trout and bind the trout liver estrogen receptor (ER). Objectives: The primary objective of this study was to determine whether multiple PFAAs enhance hepatic tumorigenesis in trout, an animal model that represents human insensitivity to peroxisome proliferators. Methods: A two-stage chemical carcinogenesis model was employed in trout to evaluate PFOA, perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluorooctane sulfonate (PFOS) and 8:2 fluorotelomer alcohol (8:2FtOH) as complete carcinogens or promoters of aflatoxin B1 (AFB1)- and/or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced liver cancer. A custom trout DNA microarray was used to assess hepatic transcriptional response to these dietary treatments in comparison to E2 and the classic peroxisome proliferator clofibrate (CLOF). Results: Incidence, multiplicity and size of liver tumors in trout fed diets containing E2, PFOA, PFNA and PFDA were significantly higher compared to AFB1-initiated animals fed control diet, whereas PFOS caused a minor increase in liver tumor incidence. E2 and PFOA also enhanced MNNG-initiated hepatocarcinogenesis. Pearson correlation analyses, unsupervised hierarchical clustering and principal components analyses showed that the hepatic gene expression profiles for E2 and PFOA, PFNA, PFDA and PFOS were overall highly similar, though distinct patterns of gene expression were evident for each treatment, particularly for PFNA. Conclusions: Overall, these data suggest that multiple PFAAs can promote liver cancer and that the mechanism of promotion may be similar to that for E2. A total of 40 samples were analyzed using a a dye-swap, reference sample hybridization protocol. Rainbow trout were exposed to the following experimental treatments via the diet for two weeks (number in parenthesis is assigned group #): (1) Control; (2) 5 mg/kg diet estradiol (E2); (3) 2000 mg/kg diet perfluorooctanoic acid (PFOA); (4) 2000 mg/kg diet perfluorononanoic acid (PFNA); (5) 2000 mg/kg diet perfluorodecanoic acid (PFDA); (6) 200 mg/kg diet perfluorooctane sulfonate; (7) 2000 mg/kg 8:2 fluorotelomer alcohol (FTOH); and (8) 2000 mg/kg diet clofibrate. A total of 40 total hepatic mRNA samples were analyzed using a dye-swap, reference sample hybridization protocol. A reference RNA pool was made by combining equal amounts of RNA from all control RNA samples, with one exception. A separate time-matched reference pool was utilized for group 6 samples (PFOS treatment). Five hybridizations were performed for each treatment group in the following pattern: Replicate A Cy5/Reference Cy3; Replicate A Cy3/Reference Cy 5; Replicate B Cy5/Reference Cy3; Replicate B Cy3/Reference Cy5; Replicate C Cy5/Reference Cy3. Thus, the experiment consisted of three biological replicates, for two of which were replicated technically.

ORGANISM(S): Oncorhynchus mykiss

SUBMITTER: Abby Benninghoff

PROVIDER: E-GEOD-31085 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project 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).

Project description:Perfluorooctane sulfonate (PFOS) is a perfluoroalkyl acid (PFAA) and a persistent environmental contaminant found in the tissues of humans and wildlife. Although blood levels of PFOS have begun to decline, health concerns remain because of the long half-life of PFOS in humans. Like other PFAAs, such as perfluorooctanoic acid (PFOA), PFOS is an activator of peroxisome proliferator-activated receptor-alpha (PPARα) and exhibits hepatocarcinogenic potential in rodents. PFOS is also a developmental toxicant in rodents where, unlike PFOA, it’s mode of action is independent of PPARα. Wild-type (WT) and PPARα-null (Null) mice were dosed with 0, 3, or 10 mg/kg/day PFOS for 7 days. Animals were euthanized, livers weighed, and liver samples collected for histology and preparation of total RNA. Gene profiling was conducted using Affymetrix 430_2 microarrays. In WT mice, PFOS induced changes that were characteristic of PPARα transactivation including regulation of genes associated with lipid metabolism, peroxisome biogenesis, proteasome activation, and inflammation. PPARα-independent changes were indicated in both WT and Null mice by altered expression of genes related to lipid metabolism, inflammation, and xenobiotic metabolism. Such results are similar to prior studies done with PFOA and are consistent with modest activation of the constitutive androstane receptor (CAR) and possibly PPARγ and/or PPARβ/δ. Unique treatment-related effects were also found in Null mice including altered expression of genes associated with ribosome biogenesis, oxidative phosphorylation and cholesterol biosynthesis. Of interest was up-regulation of Cyp7a1, a gene which is under the control of various transcription regulators. Hence, in addition to its ability to modestly activate PPARα, PFOS induces a variety of “off-target” effects as well. PPARalpha-null and wild-type male mice at 6-9 months of age were dosed by gavage for 7 consecutive days with either 0, 3, or 10 mg/kg PFOS (potassium salt) in 0.5% Tween 20. Five biological replicates consisting of individual animals were included in each dosage group. Data were compared to results previously published by our group for PFOA and Wy-14,643, a commonly used agonist of PPARalpha (Rosen et al., Toxicol Pathol. 36:592-607, 2008; GSE9796)

Project description:Beneficial effects of a soy diet on bone quality have been assumed to be due to the putative estrogenic actions of isoflavones. We studied the effects of soy protein isolate (SPI) on bone quality and compared these effects to 17Î²-estradiol (E2) in pre-pubertal rats. Female rats were weaned to a control diet with or without E2 (0.1, 1, 10 Âµg/kg/d), or SPI-containing diet with or without E2 (10 Âµg/kg/d) for 14 days beginning on postnatal day 20. In long bones from SPI-fed rats, only cancellous bone mineral density (BMD) was increased (p<0.05), while cortical BMD was decreased accompanied by lower bone strength compared to control casein-fed rats (p<0.05). In sharp contrast, 10 Âµg/kg/d E2 not only increased trabecular BMD, but also cortical BMD compared to controls. Rats treated with the combination of SPI and E2 had an intermediate bone effect. SPI increased while E2 decreased bone turnover, and increased trabecular BMD by both E2 and SPI was associated with decreased serum sclerostin levels. Microarray analysis revealed 652 genes regulated by SPI diet, 491 genes regulated by E2, and 266 genes regulated in common by both SPI diet and E2 compared to rats fed casein. The expression of caveolin-1, a protein localized in cell membrane, was down-regulated (p<0.05) in rats fed SPI, but not by E2 compared to rats fed casein. Down-regulated caveolin-1 by SPI was associated with increased BMP2, Smad and Runx2 expression in bone and osteoblasts (p<0.05). These results suggest SPI consumption results in significant non-classical estrogenic stimulation of cancellous bone formation prior to puberty, but may have adverse effects on overall bone quality and strength at this developmental stage as a result of reduced cortical bone formation. The study was designed to compare expression of bone development associated genes between soy protein isolate fed and estrogen treated pre pubertal female rats. Therefore, total RNA was isolated from bone from rats fed with control casein diet, SPI diet, 17 beta estrodial treated and SPI diet plus 17 beta estrodial.

Project description:Perfluorooctanesulfonate (PFOS) has been widely used in a variety of industrial and commercial applications as a surfactant and stain repellent. PFOS causes liver damage (including liver tumors) in experimental animals, primarily via interaction with PPARa and CAR/PXR. We investigated the involvement of microRNAs (miRNAs) in PFOS-induced hepatotoxicity, and mechanisms involved in abnormal TH homeostasis, in the livers of adult male rats exposed in feed to 50 mg PFOS/kg diet for 28 days. PFOS-treated rats exhibited expected histopathological and clinical chemistry changes. Global gene expression changes were consistent with the involvement of PPARα and CAR/PXR in PFOS-induced effects. Thirty-eight miRNAs were significantly altered. Three members of the miR-200 family were the most increased, while miR-122 and miR-21 were the most decreased, in PFOS-treated relative to control rats. Expression of the miR-23b/27b/24 cluster also decreased in PFOS-treated animals. Pathway analysis of miRNAs and associated gene expression changes demonstrated enrichment of transcripts involved in epithelial to mesenchymal transition (EMT), which is a primary process involved in tumor cell motility and cancer metastasis. Liver expression analysis revealed transcripts that may mediate PFOS effects on thyroid hormone (TH) homeostasis including: activation of the CAR/PXR pathway, phase II/III enzymes, and deiodinase. These changes are consistent with low serum TH due to enhanced metabolic clearance of TH. However, most TH hepatic target genes were not altered in a manner consistent with reduced TH signalling; suggesting that PFOS exposure did not induce functional hypothyroidism. Collectively, PFOS-induced miRNA perturbations were strongly associated with EMT suggesting an important role for miRNAs in PFOS-induced hepatotoxicity. The work also provides novel insights into the effects of PFOS on TH homeostasis. Four RNA samples from control and four from PFOS treated rats were labelled with Cyanine 5-CTP (Cy5) using Low Input Quick Amp Labelling kits (Agilent Technologies Inc.) following the manufacturer’s instruction. Universal rat reference total RNA (Agilent Technologies Inc.) was labelled with Cyanine 3-CTP (Cy3). Cy5-sample cRNA and Cy3-reference cRNA were hybridized to Agilent G4853A SurePrint G3 Rat GE 8 X 60K microarrays (Agilent Technologies Inc.) at 65°C overnight with Agilent hybridization solution. Slides were washed and scanned on an Agilent G2505B microarray scanner at 5 μm resolution, and the data were acquired with Agilent Feature Extraction software version 10.7.3.1. Microarray data quality was confirmed using Agilent Feature Extraction quality control metrics and in-house metrics (boxplots, cluster analyses, and MA plots to identify poor quality outlier arrays). All samples passed the quality control tests and were used for subsequent analyses.

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Transcript profiles of PFAAs in trout liver

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