Toxicogenomic Differences Between Nonylphenol and PCB-77 Induced Gene Signalling Pathways in Salmon Hepatocytes

In the natural environment, chemicals occur as complex mixtures whereby the biological effect of an individual chemical may be enhanced or antagonized by the presence of another chemical. We have exploited the hypothesis generating quality of suppression subtractive hybridization (SSH) as diagnostic tool to assess qualitative gene expression changes, and subsequent quantitative approach using real-time PCR in primary culture of salmon hepatocytes. The SSH method showed a total of 350 gene clones generated from a pool of salmon liver exposed to estrogen receptor agonists (NP: 100 mg/kg fish and estradiol-17b: 5 mg/kg fish) and AhR-agonists (PCB-77: 1 mg/kg and BNF: 50 mg/kg) with unique expression patterns in the forward and reverse (up- or down-regulated) subtractions. The genes consisted of representative and distinct genes distributed into several important functional categories. Based on sequence match significance and frequencies within forward and reverse libraries, these genes were spotted on a nylon membrane (SalArray) and used in a mechanistic approach for screening for the interaction between NP and PCB-77. In the experimental setup, two separate experiments were performed to study the effects of different concentration of PCB-77 and time factor, respectively. Firstly, primary salmon hepatocytes were exposed to nonylphenol (NP: 5 mM) singly or in combination with 0.001, 0.01 and 1 med mer PCB-77 and sampled at 48 h post-exposure. Secondly, hepatocytes were exposed to NP (5 mM) or PCB-77 (1 mM) singly or in combination for 12, 24, 48 and 72 h. Samples were analyzed using a validated real-time PCR for genes in the ER-pathway or known to be NP-responsive and AhR pathway or known to be PCB-77 responsive. Our data showed a reciprocal inhibitory interaction between NP and PCB-77. PCB-77 produced anti-NP-mediated effect by decreasing the mRNA expression of ER-responsive genes. NP showed anti-AhR effect or as inhibitor of AhRa, AhRR, ARNT, CYP1A1 and UDPGT expression. Interestingly, PCB-77 increased ERb mRNA expression above control and equal to NP levels. In general, we observed that NP down-regulation of salmon AhR-mediated responses appear to involve ERs. In contrast, PCB-77 down-regulation of ER-mediated responses does not appear to directly involve AhRs, as no direct relationships between these effects were observed. Overall, the findings in the present study demonstrate a complex mode of ER-AhR interaction, indicating that PCB-77-mediated antiestrogenicity may involve a possible ER-AhR competition for common co-factors. This complex mode of interaction is further supported by the effect of PCB-77 on ERb (shown as increase in transcription) with no subsequent activation of estrogen-mediated responses. This complex interaction between two different classes of ligand-activated receptors provides novel mechanistic insights on signalling pathways. Therefore, the degree of simultaneous interactions between the ER and AhR gene transcripts demonstrated in this study supports the concept of cross-talk between these signalling pathways.