Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Sensitive and robust gene expression changes in fish exposed to estrogen - a microarray approach

ABSTRACT: Background
Vitellogenin is a well established biomarker for estrogenic exposure in fish. However, effects on gonadal differentiation at concentrations of estrogen not sufficient to give rise to a measurable vitellogenin response suggest that more sensitive biomarkers would be useful. Induction of zona pellucida genes may be more sensitive but their specificities are not as clear. The objective of this study was to find additional sensitive and robust candidate biomarkers of estrogenic exposure.
Results
Hepatic mRNA expression profiles were characterized in juvenile rainbow trout exposed to a measured concentration of 0.87 and 10ng ethinylestradiol/L using a salmonid cDNA microarray. The higher concentration was used to guide the subsequent identification of generally more subtle responses at the low concentration not sufficient to induce vitellogenin. A meta-analysis was performed with data from the present study and three similar microarray studies using different fish species and platforms. Within the generated list of presumably robust responses, several well-known estrogen-regulated genes were identified. Two genes, confirmed by quantitative RT-PCR (qPCR), fulfilled both the criteria of high sensitivity and robustness; the induction of the genes encoding zona pellucida protein 3 and a nucleoside diphosphate kinase (nm23).
Conclusions
The cross-species, cross-platform meta-analysis correctly identified several robust responses. This adds confidence to our approach used for identifying candidate biomarkers. Specifically, we propose that analyses of nm23 together with zona pellucida genes may increase the possibilities to detect an exposure to low levels of estrogenic compounds in fish.

ORGANISM(S): Oncorhynchus mykiss

SUBMITTER: Lina Gunnarsson

PROVIDER: E-MEXP-1149 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Two decades after the introduction of oil-based vaccines in the control of bacterial and viral diseases in farmed salmonids, the mechanisms of induced side effects manifested as intra-abdominal granulomas remain unresolved. Side effects have been associated with generation of auto-antibodies and autoimmunity but underlying profile of the inflammatory and immune response has not been characterized. This study was undertaken with the aim to elucidate the inflammatory and immune mechanisms of granuloma formation at gene expression level associated with high and low side effect (granuloma) indices. Groups of Atlantic salmon parr were injected intraperitoneally with oil-adjuvanted vaccines containing either high or low concentrations of Aeromonas salmonicida or Moritella viscose antigens in order to induce polarized (severe and mild) granulomatous reactions. The established granulomatous reactions were confirmed by gross and histological methods at 3 months post vaccination when responses were known to have matured. The corresponding gene expression patterns in the head kidneys were profiled using salmonid cDNA microarrays followed by validation by real-time quantitative PCR (qPCR). qPCR was also used to examine the expression of additional genes known to be important in the adaptive immune response. Granulomatous lesions were observed in all vaccinated fish. Presence of severe granulomas were associated with a profile of up-regulation of innate immunity-related genes such as complement factors C1q and C6, mannose binding protein, lysozyme C, C-type lectin receptor, CD209, Cathepsin D, CD63, LECT-2, CC chemokine and metallothionein. In addition, IL-17A (Th17) was significantly up-regulated (p=0.009) in the group with severe granulomas as were arginase and IgM. None of the genes directly reflective of Th1 T cell lineage (IFN-γ, CD4) or Th2 (GATA-3) differentiation were differentially expressed. Granulomatous reactions following vaccination with oil-based vaccines in Atlantic salmon has the profile of strong expression of genes related to innate immune responses. The expression of IL-17A suggests an involvement of Th17 T cell lineage and is in conformity with strong infiltration of neutrophils and macrophages into inflamed areas. Arginase upregulation shows that macrophages in these reactions are alternatively activated, indicating a Th2-bias. To what extent the IL-17A profile reflects an autoimmune vaccine-based reaction remains elusive but would be in conformity with previous observations of autoimmune reactions in salmon vaccinated with oil-based vaccines. RNA from 12 head kidney samples (12 fish) per group were pooled into groups of 4. The RNA was amplified prior to hybridization to arrays. Three biological and 2 technical replicates (dye-swapped) were utilized in different combinations to hybridize to 18 arrays using RNA from fish with the least injection-site inflammatory reactions (FO-1) as the reference group. The assignment of microarrays to treatment groups for hybridization was randomised by using a random number generator. To minimize technical variability, target synthesis was done in batches of 6 where all treatment groups were equally represented.

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Sensitive and robust gene expression changes in fish exposed to estrogen - a microarray approach

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