Aquaporin water channnels and osmoregulation in the European eel (Anguilla anguilla): the potential toxic effects of brominated flame retardants.

Lead Research Organisation: University of Sheffield
Department Name: Biomedical Science


The regulation of water transport is fundamental to the survival of all forms of life on the planet, from simple uni-cellular organisms to complex multi-cellular plants and animals, including mammals and man. The evolutionary success and survival of aquatic organisms in many diverse freshwater (FW) and sea water (SW) habitats has also been made possible by the development of complex inter-related ion and water transport systems which allow animals and plants to osmoregulate and survive in extreme hypotonic and hypertonic environments. Well-studied examples of this are the euryhaline teleosts, such as the European eel, which exhibit the genetic plasticity to enable survival in both FW and SW environments with only minimal changes in the osmolality and ionic composition of their body fluids. In order to accomplish this, euryhaline teleosts have evolved the capacity to adapt their osmoregulatory strategies to allow the excretion of excess water and the scavenging of salts from ion-poor FW habitats while reversing these functions when entering SW environments. Central to this process is the critical function of members of a large family of membrane transporting proteins called aquaporins, that not only regulate water transport between various compartments within the body but are also essential to the maintenance of the equilibrium between total body fluid volume and water availability in the external environment. In addition to water, a subset of these proteins are also able to transport small polar solutes including glycerol and urea as well as certain gases such as carbon dioxide and ammonia, which extends their physiological roles within all organisms. All aquatic organisms, including the euryhaline eel, depend on the normal functioning of these proteins for survival in both FW and SW environments but the varied roles of these proteins, and how they are hormonally regulated are poorly understood. This research programme will determine what members of the aquaporin family are present in the osmoregulatory and reproductive tissues of the eel and will characterise changes in their abundance and function as fish move between FW and SW environments. These investigations will highlight the critical physiological roles played by the aquaporins in the maintenance of body fluid composition when eels are exposed to the changes in environmental salinity normally associated with their migration back to the Sargasso sea to breed. The study will also highlight changes in aquaporin function within the developing gonads of the sexually maturing eels, where it has recently been reported in mammals that changes in water transport within the sperm and egg are essential pre-requisites for normal reproductive development. Over the last 30 years there has been a dramatic world-wide decline in eel populations, with reports of populations in some areas dropping to less than 10% of that recorded in the late 60s and early 70's. This period of population decline exhibits an inverse relationship to the increasing production and appearance within various environmental sediments and effluents, of a group of chemicals collectively known as brominated flame retardants (BFRs). These chemicals are extremely long-lived in the environment and are know to be accumulating within different species in various food chains, all the way up to and including man. Recent reports indicate that relatively low levels of some BFRs can act as endocrine disruptors by functioning as hormone mimetics or antagonists, compromising the normal functions of the thyroid hormones and sex steroids. As part of this project we will investigate the potential effects of BFRs on the hormonal regulation of aquaporin function in the European eel as it is possible that deleterious effects of these toxins on these water transporters may compromise the successful migration of fish from FW to SW environments and/or reduce the reproductive fecundity of fish returning to the Sargasso sea.
Description Cloning, sequencing and expression (objectives 1 and 5). Full-length cDNAs were amplified, cloned and sequenced for 12 eel AQP genes (one AQP0, two AQP1s (a, b), one AQP3, two AQP4s (a, b), one AQP7, two AQP8s (a,b), two AQP9s (a, b) and one AQP10) and 8 other genes associated with either osmoregulation or sex determination. One particularly novel finding was the discovery of two AQP4 isoforms that exhibit different expression in eel tissues. A number of N- and C-terminal spliceforms have been identified which appear to be expressed in a tissue-specific manner. Homology between the eel isoforms is generally low and characteristic of that found in other species with highest homologies around the canonical NPA motifs and lowest at both N- and C-terminal domains. Expression of all isoforms was determined in a number of tissues from FW- and SW- acclimated fish. Table 1 in Appendix 1 summarises the relative general expression levels of all eel AQP isoforms across all adult developmental stages (yellow and silver), environment types (FW and SW) and the effects of infusions of the brominated flame retardants (BFRs), PBDE, HCBD or TBBPA. Treatments of silver eels over a 10 day period with BFRs resulted in tissue levels of the compounds increasing from non-detectable levels to values equivalent to that found in fish captured from heavily polluted environments. The expression levels of AQP1b in the kidney and AQP8a in the intestine exhibited very high variability, some fish exhibiting very high expression whereas others had very low, or even undetectable expression levels. Although not statistically significant due to the high inter-fish variability in the control groups, all fish exposed to any BFR exhibited virtually no AQP1b expression in the renal kidney, whereas the highly variable AQP1b expression remained in all other tissues. FW/SW transfer, the yellow silver eel transition or 10 day exposure to BFRs, failed to induce any significant changes in any AQP isoform expression in the gonads.
Subcellular locations within osmoregulatory tissues (Objective 2). Immuno-histochemical analyses identified AQP1a within blood vessel endothelial cells, the apical brush border of intestinal enterocytes (especially in the posterior intestine and rectal regions) and on the apical surfaces of a subset (possibly proximal tubules) within the renal kidney. AQP3 immunoreactivity was evident within the basolateral membranes and extensive tubular systems of the mitochondrial-rich "chloride cells", the plasma membranes of epithelial cells and epithelial progenitor cells within the stratified squamous epithelium of the gill and in mucous cells and macrophage-like cells within the distal/rectal end of the intestinal epithelium. Despite several attempts we were unable to raise isoform specific antisera to AQPs 1b and 10 and antibodies raised to AQPs 8a and 8b had poor affinity. AQP8a immunoreactivity was predominantly located within the apical brush border and to a lesser extent the cytosol of the columnar enterocytes that were present along the entire length of the intestine and into the rectum, whereas AQP8b immunoreactivity was located intracellularly in single cells found deep within the intestinal epithelium that resembled entero-endocrine cells. A subset of these cells co-stained with antibodies against serotonin. Unfortunately the affinity-purified AQP8a and 8b antibodies not only bound to proteins of the expected molecular weight for AQPs but were found to cross-react with similar affinities to other unknown higher molecular weight proteins on Western blots.
Hormones and expression (Objectives 3 and 6). Cortisol was the only hormone found to alter the expression of any AQP isoform. Thyroid hormones, estradiol and testosterone were without effect. Statistically significant increases and decreases in AQP1a isoform mRNA and protein expression were found in the intestine and kidney respectively, but only in yellow eels. Cortisol also significantly reduced mRNA and protein expression of AQP3 in the gills of yellow and silver eels. No significant change in expression of any other AQP isoform was found following any hormone infusion.
Functional characteristics of recombinant AQPs (Objectives 4 and 5). AQP1 exhibited transport of water, carbon dioxide and ammonia; AQP3 exhibited only glycerol / urea transport, AQP8a exhibited water and ammonia transport and AQP8b allowed the transport of water and glycerol/urea. AQPs 1a, 8a and 8b (all contain Cys180 in second NPA motif) were sensitive to mercury, whereas AQP3 (Ala180) was not. AQP3 was found to be sensitive to external pH and diethyl pyrocarbonate (DEPC) indicating the presence of a functionally active histidine (probably His53) residue somewhere in the sequence. Transfection of AQP3 into the mammalian renal cell line MDCK, resulted in AQP3 expression in both apical and basolateral membranes and an increase in a PKC-dependent trans-epithelial urea transport. Inhibition of PKC resulted in internalisation of AQP and reduction in urea transport. TBBPA administration failed to consistently affect water/solute transport into AQP transfected oocytes. However, both PBDE and HCBD suppressed AQP3-mediated urea uptake into oocytes in a pH-dependent manner and also disrupted the PKC-dependent trafficking of eel AQP3 in MDCK cells. PBDE inhibited translocation of AQP3 to the apical surface, whereas HBCD stimulated apical translocation but inhibited basolateral translocation. With both BFRs, substantial AQP3 was also retained within cytoplasmic vesicles. The increased apical translocation of AQP3 by HCBD was accompanied by an increase in urea uptake in polarised cell monolayers, although this was pH sensitive with activation being reversed at alkaline pH.
Exploitation Route Future research could focus on a molecular study on the metabolism of BFRs and the production of any modified metabolites by the fish. Also research on the mechanism of transcriptional control of the AQPs and the routes for mRNA degradation might determine the mode of action of BFRs in altering AQP expression.
Sectors Agriculture, Food and Drink,Environment,Other