Molecular and cellular detection of expression of vitellogenin and <Emphasis Type="Italic">zona radiata</Emphasis> protein in liver and skin of juvenile salmon (<Emphasis Type="Italic">Salmo salar</Emphasis>) exposed to nonylphenol

In developing bioassays for estrogenic effects, vitellogenin (Vtg) induction and zona radiata protein (Zr-protein) induction in males and juveniles of oviparous vertebrates have been used as sensitive biomarkers for estrogenicity. Nonylphenol (NP) produces similar and parallel expression patterns of Vtg and Zr-protein levels in plasma and surface mucus of salmon, the response being concentration- and time-dependent. We have explored the potential mechanisms of Vtg and Zr-protein expression in surface mucus by comparative molecular and cellular approaches. Liver, skin, blood, and surface mucus samples were collected from fish exposed to a single waterborne concentration of NP (10 and 60 μg/l), 3, 7, and 10 days post-exposure, for gene expression analysis (liver and skin; quantitative real-time polymerase chain reaction) and protein analysis (blood and surface mucus; enzyme-linked immunosorbent assay). Protein expression was localized by immunohistochemistry. NP produced concentration- and time-dependent increases of hepatic estrogen receptors (ERα and ERβ), Vtg, and Zr-protein mRNA and plasma protein levels. These responses paralleled cellular detection of Vtg and Zr-protein in the liver with unique expression patterns in the cytoplasm of hepatocytes, hepatic sinusoids, and endothelial cells. ERα, Vtg, and Zr-protein mRNA were detectable in the skin. ERβ was the only skin response that was NP-concentration-dependent, especially at day 10 post-exposure. Immunohistochemistry for Vtg and Zr-protein in skin showed unique expression patterns in mucus vacuoles, epidermal cells, and scales in an NP-concentration- and time-specific manner. Thus, analysis of skin mRNA levels for xenoestrogen biomarker responses is a less-promising approach than protein analysis. The immunohistochemical localization of Vtg and Zr-protein levels in the skin further validates surface mucus as a sensitive biomarker source for estrogenic compounds. These responses represent an improvement for the detection of endocrine-disrupting compounds and related pollutants in the environment. The Norwegian Research Council (NFR) financially supported this study.