Final Report Summary - METALTRANSP (Characterization of the trace metals transport and interaction mechanisms in zebrafish Danio rerio using molecular and stable isotope approaches)
Understanding the environmental toxicology of metals is essential for setting adequate site specific water quality criteria. Due to anthropogenic activities, concentrations of both essential and non-essential elements in surface waters are often far above the natural levels posing a threat to the health of aquatic organisms. At present, the Biotic Ligand Model (BLM) is the most widely used computational concept used to predict the effects of water chemistry on metal bioavailability and toxicity. However, currently these models are developed for single metals and do not take into account interactions between metals at the gill surface that affect their accumulation. In real environments metals are rarely seen in isolation and therefore a greater understanding of the uptake mechanisms is required for the development of a multimetal BLMs to avoid over- or underestimation of trace metal toxicity.
The global objective of the project was to identify shared cellular transporters in zebrafish gills that are involved in the uptake of metals from a complex environment using stable isotopes and molecular approaches. The study was designed to investigate interactions between toxic and essential metals using 106Cd-65Cu, 65Cu-204Pb and 204Pb-67Zn metal combinations. Metal accumulation rates were compared with changes in the transcript abundance of genes encoding Cu (CTR1, ATP7a), Ca (ECAC), divalent metals (DMT1) and Zn (ZIP3, ZIP7, ZIP8, ZIP10) transporting proteins.
106Cd-65Cu exposure
Experiments were conducted in water containing 2 mM CaCl2·2H2O, 0.5 mM NaHCO3, 0.5 mM MgSO4·7H2O and 0.077 mM KCl dissolved in deionised water. The test medium was spiked with: a) 0.003µM 106Cd, b) 0.025µM 106Cd, c) 0.05µM 65Cu, d) 0.5µM 65Cu, e) 0.025µM 106Cd + 0.05µM 65Cu and f) 0.025µM 106Cd + 0.5µM 65Cu. Results showed that addition of 65Cu clearly suppressed 106Cd uptake, which is in accordance with previous findings. The effect of 106Cd on 65Cu was more complex, but statistically insignificant. Molecular analysis showed a complex dynamic genetic response in the gills. There was a good agreement between temporal transcript expression patterns of Cu importing (CTR1) and exporting (ATP7a) genes. At 24 hours combined Cu-Cd exposures lead to a significant increase in the CTR1 and ECaC and a suppression of ZIP8 transcript levels. At 48 hours of exposure, a significant increase in DMT1 expression levels was observed in all treatments containing Cd, however, the effect was mitigated by addition of Cu. The comparison of changes in Cd uptake rates and temporal variations in relative transcript abundances of transporting genes suggest that Cd enters a cell via ECaC and ZIP8 channels. Cu competes for Cd at these uptake sites leading to rerouting of Cd uptake via DMT1. In spite of a reduction in ECaC and Zip8 transcript abundance at 48 h in the presence of Cu, the uptake of Cd remains linear over the 48 h and an increase in transcript levels of DMT1 at this time point suggests Cd uptake likely continues via this divalent metal ion transport protein.
65Cu-204Pb exposure
Possible involvement of ECAC channel in Cu uptake may explain occurrence of Cu-Pb interactions observed in previous studies. In this set of experiments zebrafish were exposed to: a) 0.05µM 65Cu, b) 0.05µM 204Pb, c) 0.2µM 204Pb, d) 0.05µM 65Cu +0.05µM 204Pb, and e) 0.05µM 65Cu +0.2µM 204Pb in the test medium of the same composition as in Cd-Cu experiments. Results demonstrated that addition of Pb significantly increased Cu uptake rates, while Pb had a slight, statistically insignificant inhibitory effect suggesting some competitive interactions. Molecular analysis showed absence of changes in the transcript abundance of Cu or Ca transporting genes (CTR1, ATP7a or ECAC) in spite of the observed increase in Cu uptake rates in the presence of Pb. Combined exposures elevated transcription of DMT1 at 48 hours of exposure, while low Pb concentration raised ZIP8 transcript abundance at 24h of exposure. The uptake of Pb has been associated with DMT1 transporter and it is interesting to see an increase in its expression levels only in combination with Cu at the later stage of exposure. This supports the idea of a presence of an alternative non-specific uptake pathway which is activated by multiple double charged ions and is saturated within 24 hours of exposure. After that, the uptake processes is re-routed via DMT1.
67Zn -204Pb exposure
Previous experiment demonstrated presence of a shared component, presumably calcium channel, in the uptake pathway of essential (Cu) and toxic (Cd, Pb) metals; however, the genetic analysis of transcript abundance of relevant genes was inconclusive. On the other hand, molecular data also indicated possible involvement of Zn transporting proteins in the uptake of other metals. To further investigate this possibility, zebrafish were exposed to: a) 0.75µM 67Zn, b) 3.0µM 67Zn, c) 0.05µM 204Pb, d) 0.75µM 67Zn + 0.05µM 204Pb, e) 3.0µM 67Zn + 0.05µM 204Pb. In order to increase the proportion of bioavailable Pb, the composition of the exposure medium was modified by decreasing Ca content to 0.5 mM Ca.
The metal accumulation data showed that zinc uptake rates were unaffected by the addition of lead, which was not unexpected considering finely tuned Zn regulating system in the gill. On the other hand, addition of Zn at low concentration stimulated Pb uptake. At higher Zn level, the Pb uptake rates were the same as in Pb only exposure suggesting activation of a different Pb-insensitive Zn uptake pathway. Molecular analysis of gene transcripts abundance revealed significant upregulation of CTR1, ZIP7 and ZIP10 transporters at 48 hours of exposure in Pb-only treatment. The increase in CTR1 expression levels would explain elevated Cu uptake rates observed in Pb-Cu experiment, which were not apparent during that experiment, possibly due to a high Ca content of the medium. The obtained results suggest that Zn transporting system plays a role in Pb uptake process in a concentration dependent manner due to activation of different transporting proteins.
The results of the current project indicate that in real environments, where metals are rarely seen in isolation, the accumulation process may not be accurately predicted based on single metal exposure model. Essential and non-essential metals share uptake pathways that are activated depending on the concentrations of the present metals. This work will provide new information for the development of metal mixture BLM models to be used by environmental regulating agencies to more accurately estimate the toxicity level of specific aquatic environment.
The global objective of the project was to identify shared cellular transporters in zebrafish gills that are involved in the uptake of metals from a complex environment using stable isotopes and molecular approaches. The study was designed to investigate interactions between toxic and essential metals using 106Cd-65Cu, 65Cu-204Pb and 204Pb-67Zn metal combinations. Metal accumulation rates were compared with changes in the transcript abundance of genes encoding Cu (CTR1, ATP7a), Ca (ECAC), divalent metals (DMT1) and Zn (ZIP3, ZIP7, ZIP8, ZIP10) transporting proteins.
106Cd-65Cu exposure
Experiments were conducted in water containing 2 mM CaCl2·2H2O, 0.5 mM NaHCO3, 0.5 mM MgSO4·7H2O and 0.077 mM KCl dissolved in deionised water. The test medium was spiked with: a) 0.003µM 106Cd, b) 0.025µM 106Cd, c) 0.05µM 65Cu, d) 0.5µM 65Cu, e) 0.025µM 106Cd + 0.05µM 65Cu and f) 0.025µM 106Cd + 0.5µM 65Cu. Results showed that addition of 65Cu clearly suppressed 106Cd uptake, which is in accordance with previous findings. The effect of 106Cd on 65Cu was more complex, but statistically insignificant. Molecular analysis showed a complex dynamic genetic response in the gills. There was a good agreement between temporal transcript expression patterns of Cu importing (CTR1) and exporting (ATP7a) genes. At 24 hours combined Cu-Cd exposures lead to a significant increase in the CTR1 and ECaC and a suppression of ZIP8 transcript levels. At 48 hours of exposure, a significant increase in DMT1 expression levels was observed in all treatments containing Cd, however, the effect was mitigated by addition of Cu. The comparison of changes in Cd uptake rates and temporal variations in relative transcript abundances of transporting genes suggest that Cd enters a cell via ECaC and ZIP8 channels. Cu competes for Cd at these uptake sites leading to rerouting of Cd uptake via DMT1. In spite of a reduction in ECaC and Zip8 transcript abundance at 48 h in the presence of Cu, the uptake of Cd remains linear over the 48 h and an increase in transcript levels of DMT1 at this time point suggests Cd uptake likely continues via this divalent metal ion transport protein.
65Cu-204Pb exposure
Possible involvement of ECAC channel in Cu uptake may explain occurrence of Cu-Pb interactions observed in previous studies. In this set of experiments zebrafish were exposed to: a) 0.05µM 65Cu, b) 0.05µM 204Pb, c) 0.2µM 204Pb, d) 0.05µM 65Cu +0.05µM 204Pb, and e) 0.05µM 65Cu +0.2µM 204Pb in the test medium of the same composition as in Cd-Cu experiments. Results demonstrated that addition of Pb significantly increased Cu uptake rates, while Pb had a slight, statistically insignificant inhibitory effect suggesting some competitive interactions. Molecular analysis showed absence of changes in the transcript abundance of Cu or Ca transporting genes (CTR1, ATP7a or ECAC) in spite of the observed increase in Cu uptake rates in the presence of Pb. Combined exposures elevated transcription of DMT1 at 48 hours of exposure, while low Pb concentration raised ZIP8 transcript abundance at 24h of exposure. The uptake of Pb has been associated with DMT1 transporter and it is interesting to see an increase in its expression levels only in combination with Cu at the later stage of exposure. This supports the idea of a presence of an alternative non-specific uptake pathway which is activated by multiple double charged ions and is saturated within 24 hours of exposure. After that, the uptake processes is re-routed via DMT1.
67Zn -204Pb exposure
Previous experiment demonstrated presence of a shared component, presumably calcium channel, in the uptake pathway of essential (Cu) and toxic (Cd, Pb) metals; however, the genetic analysis of transcript abundance of relevant genes was inconclusive. On the other hand, molecular data also indicated possible involvement of Zn transporting proteins in the uptake of other metals. To further investigate this possibility, zebrafish were exposed to: a) 0.75µM 67Zn, b) 3.0µM 67Zn, c) 0.05µM 204Pb, d) 0.75µM 67Zn + 0.05µM 204Pb, e) 3.0µM 67Zn + 0.05µM 204Pb. In order to increase the proportion of bioavailable Pb, the composition of the exposure medium was modified by decreasing Ca content to 0.5 mM Ca.
The metal accumulation data showed that zinc uptake rates were unaffected by the addition of lead, which was not unexpected considering finely tuned Zn regulating system in the gill. On the other hand, addition of Zn at low concentration stimulated Pb uptake. At higher Zn level, the Pb uptake rates were the same as in Pb only exposure suggesting activation of a different Pb-insensitive Zn uptake pathway. Molecular analysis of gene transcripts abundance revealed significant upregulation of CTR1, ZIP7 and ZIP10 transporters at 48 hours of exposure in Pb-only treatment. The increase in CTR1 expression levels would explain elevated Cu uptake rates observed in Pb-Cu experiment, which were not apparent during that experiment, possibly due to a high Ca content of the medium. The obtained results suggest that Zn transporting system plays a role in Pb uptake process in a concentration dependent manner due to activation of different transporting proteins.
The results of the current project indicate that in real environments, where metals are rarely seen in isolation, the accumulation process may not be accurately predicted based on single metal exposure model. Essential and non-essential metals share uptake pathways that are activated depending on the concentrations of the present metals. This work will provide new information for the development of metal mixture BLM models to be used by environmental regulating agencies to more accurately estimate the toxicity level of specific aquatic environment.