The effects of short-term exposure to municipal wastewater effluent on signalling pathways in the liver of fish.

Arstikaitis, Jennifer (2013). The effects of short-term exposure to municipal wastewater effluent on signalling pathways in the liver of fish. Mémoire. Québec, Université du Québec, Institut National de la Recherche Scientifique, Maîtrise en sciences expérimentales de la santé, 137 p.

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Résumé

Characterization of Montreal municipal wastewater effluent (MWWE) has identified the presence of pharmaceuticals and persona} care products (PPCP), alkyphenols (AP), polychlminated biphenyls (PCB), polyaromatic hydrocarbons (PAH), and heavy metals. MWWE contains mixtures of xenobiotics that have been shown to disrupt normal metabolic and cellular functions. Previous studies have shown that fish exposed to MWWE have resulted in altered cellular signaling in the testis. These processes are facilitated by transmembrane proteins called connexins (Cx) which form gap junctions (GJ). GJ allow the passage of small molecules between neighbouring cells, called gap junction intercellular signaling (GJIC). Brook trout exposed to MWWE for 4 and 12 weeks showed altered testicular Cx levels at both ti me points. At 4 weeks there was a significant increase in both Cx43 and Cx31. At 12 weeks a decrease in Cx31 and an increase in Cx43 and Cx43.4 was observed. The presence of xenobiotics has been shown to target and disrupt Cx mRNA levels resulting in altered cellular functions. Similar results were reported in Spottail shiners living within the effluent plume of MWWE. Shiners displayed altered testicular Cx levels resulting in delayed spermatogenesis, increased vitellogenin levels, intersex characteristics and decreased sperm motility. MWWE has been shown to target Cxs in the testis causing an alteration in key signaling pathways. lnterestingly, few studies have examined the effects of MWWE on cellular signaling in the liver considering the important role it plays in xenobiotic metabolism. Xenobiotics can accumulate in the li ver if the complex signaling required to carry out the detoxification process is disrupted. GJIC plays an important role in the regulation of normal homeostatic processes in the liver including xenobiotic metabolism, bile secretion and glycogenolysis. Fish exposed to MWWE have demonstrated altered Cx expression levels and cellular signaling in the testis, it can be hypothesized that a similar disruption of GJIC could occur in the liver. There are presently no studies that have examined the effects of MWWE on the liver and currently only one Cx has been identified in the Iiver of fish, Cx43 in the zebrafish. ln the present study two Cxs were identified in the li ver of the brook trout, Cx43 and Cx30. When compared to mammalian Cxs, brook trout Cx43 was partially homologous to GJAl in the mouse and rat. Brook trout Cx30 shared sorne homology to mouse GJB6 and human Cx26. This is the first report of Cx30 in the liver of fish. After exposing brook trout for 12 weeks to MWWE, a large increase in Cx30 mRNA levels was observed at the two highest effluent concentrations. Similar results have been previously repmted in the testis and were shown to disrupt normal GJIC. There were no changes to Cx43 levels suggesting either individual contaminants targeting Cx43 were not present or the exposure period was too short for accumulation to occur. To further investigate the effects of MWWE on key signaling pathways in the liver, juvenile fathead minnow were exposed to 20 % MWWE for 21 days. This study aimed to identify key signaling pathways affected by toxicants contained within the effluent mixture. Using a 15 208 eDNA microarray and pathway analysis software, three groups of genes, organized by function, were identified as being altered suggesting effects on various signaling pathways. These groups include genes involved in cell adhesion, inflammation and kinases. As well, two key signaling pathways were identified as having a large number of genes affected, including the anti­ estrogenic and Wnt signaling pathways. The anti-estrogenic and Wnt signaling pathways are critical during fish development, alterations have been shown to cause developmental malformations, cancer and are implicated in a number of pathologies. The present study found that over ten genes were altered in these pathways suggesting a disruption in normal developmental processes. Thirteen genes involved in the anti­ estrogenic pathway were altered and suggested an overall inhibition of the pathway further confirming previous studies that found a long-term (12 weeks) exposure was required to measure an induction of estrogenic effects. These results also suggest that a short-term exposure may elicit an initial inhibitory effect of estrogen. The Wnt signaling pathway is involved in cell-to-cell signaling during key developmental periods including embryogenesis. Thirteen genes involved in the Wnt signaling pathway were altered including the down-regulation of Wnt genes required for the activation of the pathway and the up-regulation of Wnt signaling inhibitors. These findings suggest that a short-term exposure to MWWE can result in the inhibition of the Wnt signaling pathway which plays a critical role during development. Lastly, to assess whether the inhibition of the Wnt signaling pathway was detected at the protein leve) a western blot was carried out to measure and compare beta-catenin (CTNNB1) levels. A small decrease in nuclear CTNNB1 levels was observed in exposed fish but when statistically compared to cytosolic protein levels this difference was not signficiant. Overall, this study identified two Cxs in the liver of the brook trout and showed that Cx levels can be altered by the xenobiotics readily found in MWWE. The use of microatTay technology allowed for thousands of genes to be studied in parallel and to identify three groups of genes and two key signaling pathways that were altered after a short-term exposure to MWWE.

Type de document:	Thèse Mémoire
Directeur de mémoire/thèse:	Cyr, Daniel G.
Mots-clés libres:	eau use ; Montreal ; foie ; omble-de-fontaine ; metal ; truite ; effluent ; poisson mene-a-grosse-tete ; alhyphenol ; biphenol ; polychlore ; hydrocarbure ; polycyclique ; aromatique
Centre:	Centre INRS-Institut Armand Frappier
Date de dépôt:	06 nov. 2015 16:37
Dernière modification:	06 nov. 2015 16:37
URI:	https://espace.inrs.ca/id/eprint/2355

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