Unknown,Transcriptomics,Genomics,Proteomics

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Mechanism for hepatic tumor promotion by PFOA in rainbow trout

ABSTRACT: 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).

ORGANISM(S): Oncorhynchus mykiss

SUBMITTER: Susan Tilton

PROVIDER: E-GEOD-7837 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description: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.

Project description:17alpha-ethinylestradiol (EE2) is one of the most potent estrogens that have the ability to interfere with the endocrine system of fish. The objective was to investigate the effects and mechanisms of action caused by 60 days of dietary exposure to 0.2 mg EE2/kg and 0.07 mg EE2/kg feed in female largemouth bass (LMB) during the reproductive season. Microarrays and pathway analyses were performed on hepatic tissues to identify genes and biological processes altered in female LMB by EE2 exposure. The hypothesis was that the two concentrations of EE2 would produce dose-response changes in sensitive genes. Body and ovary weights were measured and blood was collected for measurement of plasma steroid hormones (17beta-estradiol (E2), testosterone (T)) and vitellogenin (VTG) using ELISA. The 0.2 mg EE2/kg feed exposure reduced the gonadosomatic index (GSI) by 75%, and plasma levels of E2 and T were reduced by over 90%. Plasma VTG was increased by approximately 100% (from 4 to 8mg/ml) by the 0.2 mg/kg treatment. T levels, from the 0.07 mg EE2/kg feed, reduced GSI by approximately 30% and circulating E2 and T by ~80% but did not affect VTG concentrations. We found 1,594 and 1,165 genes were significantly affected (p<0.05) by the 0.07 mg EE2/kg feed and 0.2 mg EE2/kg feed, respectively. Gene ontology (GO) analysis revealed that there were different biological processes regulated by the two concentrations of EE2. Pathway analysis showed that the 0.07 mg EE2/kg feed exposure caused differential regulation of genes associated with fatty acid biosynthesis and glycolysis, indicating some metabolic effects. In contrast, the 0.2 mg EE2/kg feed exposure altered transcription of genes involved in immune response and apoptosis, suggesting a toxic response at this concentration. These results suggest that the two concentrations demonstrated distinct physiological responses, with the higher concentration inducing complete endocrine disruption in LMB. These findings demonstrate the usefulness of microarrays to identify possible biomarkers and modes of toxic action to dietary exposure in LMB. Two concentrations of EE2 would produce dose-response changes in sensitive genes. Female LMB were fed 5 days per week for 60 days with floating pellets that contained 0.07 or 0.2 mg/kg of EE2.

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Mechanism for hepatic tumor promotion by PFOA in rainbow trout

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