Expression of genes involved in copper homeostasis in a fish (Sparus aurata) exposed to waterborne and dietary copper

Copper is an essential micronutrient, but in excess it is highly toxic due to reaction with intracellular thiol groups and the initiation of oxidative damage. Recent studies in mammals have shown that cellular uptake and efflux are mediated by high affinity carriers, whilst cellular toxicity is minimised both by a series of specific chaperone proteins which carry and deliver the metal to apometalloenzymes and by sequestration of excesses by metallothionein. In aquatic organisms anthropogenic copper discharges (eg. from viniculture) are a serious problem in some areas and in this preliminary study we have investigated copper homeostasis in a copper-exposed fish, the Sea Bream, Sparus aurata. Further studies on two fish species which show an apparent defect in hepatic copper efflux have also been initiated. cDNAs for a high affinity copper uptake transporter (saCTR1), a copper chaperone (saATOX1), an efflux pump (saATP7) and the heavy metal detoxification protein metallothionein (MT) were cloned, the first three for the first time from a lower vertebrate. Quantitative assays for transcript expression were developed and used to study effects of exposure to both waterborne copper and different dietary copper levels. The deduced amino acid sequences of the four investigated protein showed very high structural conservation between fish and man. Responses to waterborne copper exposure were most marked. Expression of saCTR1mRNA was reduced by copper exposure in gill and liver, there was an initial induction of hepatic saATP7mRNA expression, and hepatic MTmRNA levels were induced, indicating regulation of intracellular copper. Moreover induction of glutathione reductase indicated that it elicited an oxidative stress response. Dietary exposure reduced CTR1 mRNA expression in intestine but at low dose levels there was an initial elevation in both CTR1 and ATP7 mRNA levels in liver. These results indicate tissue differences in response and one explanation which is being investigated is that this may be attributable to differences in copper speciation and availability. AKNOWLEDGEMENTS: Work supported by EU LSF and grant from the Ministero Politiche Agricole, VI pian