Marleen Eyckmans

Exposure to waterborne copper reveals differences in oxidative stress response in three freshwater fish species

Among species, various strategies in metal handling can occur. Moreover, the same metal concentration, or even the same metal dose, does not always seem to exert the same effect in different species. Here, we have investigated differences in a copper induced oxidative stress response between rainbow trout (Oncorhynchus mykiss), common carp (Cyprinus carpio) and gibel carp (Carassius auratus gibelio). Fish were exposed to two sub-lethal Cu concentrations, an identical concentration of 50μg/l for all fish species and an identical toxic dose which was 10% of the concentration lethal to 50% of the fish within 96h of exposure (LC50 96h value) for each of the 3 species (20μg/l for rainbow trout, 65μg/l for carp and 150μg/l for gibel carp). Different anti-oxidative enzyme (superoxide dismutase, glutathione reductase and catalase) activities and anti-oxidant (reduced glutathione and reduced ascorbate) concentrations were determined in gill samples collected after 1h, 12h, 24h, 3 days, 1 week and 1 month of Cu exposure. Changes in the measured parameters were present in all 3 species, yet a clear differentiation between fish species could be made before and during the exposure. The ascorbate levels of gibel carp were twice as high as those in common carp or rainbow trout. In contrast, the level of glutathione in rainbow trout was more than twice of that in the two other species. Also, glutathione reductase activity of rainbow trout was higher than in the other species. In rainbow trout a decrease of reduced ascorbate and reduced glutathione was observed in the beginning of the exposure, indicating that ROS scavenging molecules were under pressure. This was followed by an increase in the activity of superoxide dismutase after 3 days of exposure. In contrast, common carp and especially gibel carp enhanced their anti-oxidant enzyme activities as quickly as in the first day of exposure. Furthermore, our research seems to confirm that some fish rely more on glutathione as a first line of defence against metal exposure, while others rely more on metallothionein in combination with anti-oxidant enzymes.

Metal accumulation and metallothionein induction in the spotted dogfish **Scyliorhinus canicula**

Recent studies indicate that elasmobranch fish respond differently to metal exposure than marine teleosts. Accumulation rates can be high, which despite the fact that normal background levels for metals in the marine environment are low, is worrying due to the long life span and late fecundity of most shark. The goals of the present study were to examine differences in accumulation rates and toxicity of a range of metals at equimolar concentrations (10microM) in the Mediterranean or spotted dogfish, Scyliorhinus canicula. For this purpose, we exposed the dogfish to Ni (587microg/L), Cd (1124microg/L), Pb (2072microg/L), Cu (635microg/L), and Ag (1079microg/L and two additional exposures at 10microg/L and 1microg/L) for one week and measured total metal accumulation, metallothionein induction, and parameters related to osmoregulation. Our study confirms the high toxicity and accumulation rates of Ag for elasmobranch fish, even at levels 100 to 1000 times lower than exposure levels of other metals. Also Pb accumulated readily in all organs, but did not cause any osmoregulatory disturbance at the exposure levels used. Ni and Cd seem to accumulate primarily in the kidney while Cu mainly accumulated in liver. In contrast to Ni and Cd, the three other metals Ag, Cu and Pb accumulated in the rectal gland, an important organ for osmoregulation and possible target organ for metal toxicity. Only Cu succeeded in initiating a protective response by inducing MT synthesis in liver and gills.

Hormonal and ion regulatory response in three freshwater fish species following waterborne copper exposure.

We evaluated effects of sublethal copper exposure in 3 different freshwater fish: rainbow trout (Oncorhynchus mykiss), common carp (Cyprinus carpio) and gibel carp (Carassius auratus gibelio). In a first experiment we exposed these fishes to an equally toxic Cu dose, a Cu level 10 times lower than their 96 h LC50 value: 20, 65, and 150 microg/L Cu. In a second series we exposed them to the same Cu concentration (50 microg/L). Na+/K+-ATPase activity in gill tissue was disturbed differently in rainbow trout then in common and gibel carp. Rainbow trout showed a thorough disruption of plasma ion levels at the beginning of both exposures, whereas common carp and gibel carp displayed effects only after 3 days. Rainbow trout and common carp thyroid hormones experienced adverse effects in the beginning of the exposure. The involvement of prolactin in handling metal stress was reflected in changes of mRNA prolactin receptor concentrations in gill tissue, with an up regulation of this mRNA in rainbow trout and a down regulation in gibel carp, which was more pronounced in the latter. Overall, rainbow trout appeared more sensitive in the beginning of the exposure, however, when it overcame this first challenge, it handled copper exposure in a better manner then common and gibel carp as they showed more long term impacts of Cu exposure.

Comparative proteomics of copper exposure and toxicity in rainbow trout, common carp and gibel carp.

Species specific differences in transporters, chaperones, metal binding proteins and other targets are important in metal toxicity. Therefore, we have studied the effects of copper exposure on the proteome of gill tissue from Oncorhynchus mykiss, Cyprinus carpio and Carassius auratus gibelio, which have different sensitivities toward copper. Fish were exposed to the Flemish water quality standard for surface waters, being 50μg/L, for 3 days. Sampled gill tissue was subjected to a 2D-Dige and an iTRAQ analysis. While gibel carp showed more positive responses such as increased apolipoprotein A-I, transferrin and heat shock protein 70, common carps gill tissue on the other hand displayed a changed actin cytoskeleton, and indications of a changed metabolism. These last two traits were evident in rainbow trout as well, together with decreased expressions of transferrin and albumin. urthermore, the Weighted Gene Co-Expression Network Analysis of rainbow trout data revealed a network of 98 proteins related to Cu accumulation in gill, of which the occurrence of proteins related to oxidative stress, such as superoxide dismutase and cytochrome c were promising. Additionally, the outcome of the different proteomics techniques demonstrates the usefulness of iTRAQ analysis compared to 2D-Dige and the need for fully annotated genomes.

Are Accumulated Sulfide-Bound Metals Metabolically Available in the Benthic Oligochaete Tubifex tubifex?

The present study evaluates the relationship between metal-binding sediment characteristics like acid volatile sulfides (AVS), metal accumulation, and internal metal distribution in the benthic oligochaete Tubifex tubifex and relates this accumulation to the induction of metallothionein-like proteins (MTLPs). In total, 15 Flemish lowland rivers were sampled. Cd, Cu, Zn, Pb, Ni, As, Cr, Co, and Ag concentrations were measured in environmental fractions (water and sediment) and worm tissue (both total and subcellular fractions). Furthermore, total cytosolic MTLP concentrations were measured in the worm tissue. Our results showed that Cd, Pb, Ni, and Cr were mainly stored as biological detoxified metal (BDM) while Cu, Zn, As, and Ag were mostly available in the metal sensitive fraction (MSF). A remarkable difference in the subcellular distribution of accumulated Cd, Ni, and Co between anoxic (SEMMe-AVS<0; mostly stored as BDM) and oxic (SEMMe-AVS>0; mostly stored in the MSF) sediments was noticed. Moreover, a rapid increase in MTLP induction was found when SEMTot-AVS>0. Our results indicate that the accumulated sulfide-bound metals were detoxified and little available to the metabolism of T. tubifex under anoxic conditions.

Influence of food ration, copper exposure and exercise on the energy metabolism of common carp (Cyprinus carpio)

The present study was conducted to extend the understanding of the combined physiological effects of different food rations in combination with sublethal levels of copper in common carp (Cyprinus carpio). Fish acclimated to low (0.5% body weight) and high (5% body weight) food rations were exposed to 1 microM copper for a period of 28 days and kept for a further 14 days in copper free water to examine their recovery. Measurements of oxygen consumption, ammonia excretion and ammonia accumulation in plasma and muscle were done at various time intervals during the experimental period. Overall, oxygen consumption and ammonia excretion rates were significantly affected by food ration in both copper free and copper exposed fish. Additional challenges, such as copper exposure and/or exercise, significantly increased plasma and muscle ammonia in the fish fed a high food ration. Muscle ammonia levels in general responded slower (first increase after 72 h) and recovered within 2 weeks of exposure. There was a significant correlation between plasma ammonia levels, muscle ammonia levels and ammonia excretion rates. Influence of copper in terms of ammonia excretion and plasma ammonia accumulation was observed in high ration fish but low ration fish remained unaffected. This clearly indicates that ammonia metabolism was significantly influenced by copper in this group of fish showing that during unfavourable environmental conditions a high amount of food supply may turn deleterious to fish.

Subcellular differences in handling Cu excess in three freshwater fish species contributes greatly to their differences in sensitivity to Cu

Since changes in metal distribution among tissues and subcellular fractions can provide insights in metal toxicity and tolerance, we investigated this partitioning of Cu in gill and liver tissue of rainbow trout (Oncorhynchus mykiss), common carp (Cyprinus carpio) and gibel carp (Carassius auratus gibelio). These fish species are known to differ in their sensitivity to Cu exposure with gibel carp being the most tolerant and rainbow trout the most sensitive. After an exposure to 50 μg/l (0.79 μM) Cu for 24h, 3 days, 1 week and 1 month, gills and liver of control and exposed fish were submitted to a differential centrifugation procedure. Interestingly, there was a difference in accumulated Cu in the three fish species, even in control fishes. Where the liver of rainbow trout showed extremely high Cu concentrations under control conditions, the amount of Cu accumulated in their gills was much less than in common and gibel carp. At the subcellular level, the gills of rainbow trout appeared to distribute the additional Cu exclusively in the biologically active metal pool (BAM; contains heat-denaturable fraction and organelle fraction). A similar response could be seen in gill tissue of common carp, although the percentage of Cu in the BAM of common carp was lower compared to rainbow trout. Gill tissue of gibel carp accumulated more Cu in the biologically inactive metal pool (BIM compared to BAM; contains heat-stable fraction and metal-rich granule fraction). The liver of rainbow trout seemed much more adequate in handling the excess Cu (compared to its gills), since the storage of Cu in the BIM increased. Furthermore, the high % of Cu in the metal-rich granule fraction and heat-stable fraction in the liver of common carp and especially gibel carp together with the better Cu handling in gill tissue, pointed out the ability of the carp species to minimize the disadvantages related to Cu stress. The differences in Cu distribution at the subcellular level of gills and liver of these fish species strongly reflects their capacity to handle Cu excess and is one of the greatest contributors to their difference in sensitivity to Cu.

Physiological effects of waterborne lead exposure in spiny dogfish (Squalus acanthias )

To broaden our knowledge about the toxicity of metals in marine elasmobranchs, cannulated spiny dogfish (Squalus acanthias) were exposed to 20 μM and 100 μM lead (Pb). Since we wanted to focus on sub lethal ion-osmoregulatory and respiratory disturbances, arterial blood samples were analysed for pH(a), PaO(2), haematocrit and total CO(2) values at several time points. Plasma was used to determine urea, TMAO, lactate and ion concentrations. After 96 h, Pb concentrations were determined in a number of tissues, such as gill, rectal gland, skin and liver. To further investigate ion and osmoregulation, Na(+)/K(+)-ATPase activities in gill and rectal gland were analysed as well as rates of ammonia and urea excretion. Additionally, we studied the energy reserves in muscle and liver. Pb strongly accumulated in gills and especially in skin. Lower accumulation rates occurred in gut, kidney and rectal gland. A clear disturbance in acid-base status was observed after one day of exposure indicating a transient period of hyperventilation. The increase in pH(a) was temporary at 20 μM, but persisted at 100 μM. After 2 days, plasma Na and Cl concentrations were reduced compared to controls at 100 μM Pb and urea excretion rates were elevated. Pb caused impaired Na(+)/K(+)-ATPase activity in gills, but not in rectal gland. We conclude that spiny dogfish experienced relatively low ion-osmoregulatory and respiratory distress when exposed to lead, particularly when compared to effects of other metals such as silver. These elasmobranchs appear to be able to minimize the disturbance and maintain physiological homeostasis during an acute Pb exposure.