Gene Expression Patterns in Estrogen (Nonylphenol) and Aryl Hydrocarbon Receptor Agonists (PCB-77) Interaction Using Rainbow Trout (Oncorhynchus Mykiss) Primary Hepatocyte Culture

Abstract

It was previously reported that in vivo exposure of fish to combined aryl hydrocarbon receptor agonist (AhR; 3,3′,4,4′-tetrachlorobiphenyl,PCB-77) and estrogen receptor agonist (ER; nonylphenol. NP) resulted in potentiation and inhibition (depending on dose ratio, sequential order of exposure, and seasonal changes) of NP-induced responses by PCB-77. The experiments described in this report extend this study by testing whether the effects of PCB-77 on NP-induced ER signaling are mediated through AhR-induced transcriptional suppression of target genes. Trout hepatocytes were isolated by a two-step collagenase perfusion method. After 48-h culture, hepatocytes were exposed to 5 or 10 µM nonylphenol (NP) singly and in combination with PCB-77 at 0.1, 1, and 10 µM. Cells were harvested after 96-h exposure and processed for RNA isolation. Gene expression patterns were quantified using real-time polymerase chain reaction (PCR) with specific primer sets and by Northern blot. Exposure of cells to NP caused significant elevation of ERα, ERβ, Vtg, and Zrp mRNA expressions, while combined exposure with PCB-77 concentration inhibited NP-induced ERs and their target gene expressions. Exposure of trout hepatocytes to PCB-77 alone caused a rapid induction of cytochrome P-450 (CYP) 1A1 mRNA, and combined exposure with NP caused significant reduction in PCB-77 induced CYP1A1 gene expression. Exposure of cells to PCB-77 concentrations induced significant reduction in AhRα mRNA (except 1 µM PCB-77, which caused the induction of AhRα mRNA levels). AhRβ mRNA levels in the cells were inhibited after 96-h exposure to PCB-77, while combined exposure with 5 µM NP restored the PCB-77-inhibited AhRβ mRNA levels to baseline. Taken together, the overall results in this study show that PCB-77 suppresses the gene expression of the ERs and their target genes by transcription mechanism(s). The roles of AhRs in mediating these responses seem to involve the ligand-activated AhR transcriptional induction of CYP1A1. In addition to their frequently described functions as activators of metabolic potentiation and detoxification of various foreign chemicals, data presented in the present study point to other endogenous functions of AhRs that need to be studied further.

Notes

^a Sequences are given in the 5′–3′ order.

Bemanian, V. 2003. The aryl hydrocarbon receptor signaling pathway in Atlantic salmon: With emphasis on the cross-talk between the aryl hydrocarbon receptor and estrogen receptor α signaling pathways. PhD thesis: Department of Molecular Biology, University of Bergen, Norway.