Som Niyogi

Application of XANES spectroscopy in understanding the metabolism of selenium in isolated rainbow trout hepatocytes: insights into selenium toxicity

Selenium (Se) is an essential element, but causes toxic effects in fish at a slightly elevated level beyond the threshold. However, the degree of Se toxicity differs depending on the chemical forms of Se (e.g., organic vs. inorganic) to which fish are exposed to. The mechanisms of Se metabolism and toxicity in fish, particularly at cellular level, are poorly understood. The present study was designed to examine the metabolic fate of different seleno-compounds, both inorganic and organic, in isolated hepatocytes of rainbow trout (Oncorhynchus mykiss) in primary culture using XANES spectroscopy. In cells exposed to 100 μM of selenate and selenite for 6-24 h, elemental Se was found to be the primary metabolite. Whereas, selenocystine appeared to be the major metabolite in cells exposed to 100 μM seleno-L-methionine for 6-24 h. Interestingly, we recorded L-methionine-γ-lyase activity in S9 fraction of cell lysate-an enzyme that directly catalyzes selenomethionine into methylselenol. We also found concurrent reduction of glutathione (GSH) concentration following reaction of seleno-L-methionine with cellular S9 fraction. Moreover, we observed a rapid increase in cellular reactive oxygen species (ROS) generation with increasing seleno-L-methionine exposure dose (100-1000 μM). These findings indicated the rapid cellular metabolism of seleno-L-methionine into methylselenol at higher exposure dose (≥100 μM), and the occurrence of GSH mediated redox cycling of methylselenol--a process that is known to produce reactive oxygen species (ROS). Overall, our results suggest that inorganic and organic selenium are metabolized through different metabolic pathways in rainbow trout hepatocytes. The findings of our study have important implications for understanding the chemical species-specific differences in Se toxicity to fish.

Effects of dietary cadmium exposure on tissue-specific cadmium accumulation, iron status and expression of iron-handling and stress-inducible genes in rainbow trout: Influence of elevated dietary iron

Recent evidences suggest that dietary cadmium (Cd) uptake likely occurs via the dietary iron (Fe) uptake pathway in freshwater fish, at least in part. The present study investigated the interactive effects of dietary Cd and Fe in juvenile rainbow trout (Oncorhynchus mykiss). Fish were treated for four weeks with four different diets: normal Fe, high Fe, normal Fe plus Cd, and high Fe plus Cd. Physiological parameters, tissue-specific Fe and Cd level, plasma Fe status, and tissue-specific mRNA expression of transferrin, metallothioneins (MT-A and MT-B) and heat shock proteins 70 (HSP70a and HSP70b) were analyzed. Exposure to dietary Cd increased Cd burden in the following order: intestine>kidney>stomach>liver>gill>carcass. Interestingly, high dietary Fe reduced Cd accumulation in the stomach and intestine as well as in the wholebody of fish. Dietary Cd increased hepatic transferrin mRNA expression and total Fe binding capacity in the plasma, indicating the effect of Cd on Fe handling in fish. The mRNA expression of MTs and HSP70s was also increased in various tissues following dietary Cd exposure, however the response profile of different MT and HSP70 genes was not consistent among different tissues. In general, MT-A was more responsive to Cd exposure in the intestine and liver, whereas MT-B was more responsive in the kidney. Similarly, HSP70a expression was more sensitive to Cd exposure than HSP70b, particularly in the intestine. Interestingly, high Fe diet suppressed Cd-induced induction of transferrin, MT and HSP70 genes in various tissues. Overall, our study suggests that elevated dietary Fe can reduce Cd accumulation and ameliorate Cd-induced stress responses in freshwater fish.

The effects of chronic exposure to environmentally relevant levels of waterborne cadmium on reproductive capacity and behaviour in fathead minnows.

Cadmium (Cd) is a priority pollutant in aquatic ecosystems because of its highly toxic effects at low concentrations. Recent work has highlighted that at sublethal concentrations, Cd can impair the ability of fish to respond to important sensory cues. In the present study, we examined whether chronic exposure to environmentally relevant concentrations of waterborne Cd (1, 2.5 and 5xa0μg/L) can result in reproductive impairment in fathead minnows (Pimephales promelas) as measured by the standard Organization for Economic Cooperation and Development (OECD) 21-day reproductive assay. In addition, we also evaluated the effects of chronic waterborne Cd exposure on Cd accumulation in target tissues (liver and ovary) and plasma estradiol level in females and on the reproductive behaviour of fathead minnows. We found that the standard endpoints used in the OECD reproductive assays (e.g., egg production, number of spawning attempts, brood size) are more sensitive to Cd exposure than behavioural endpoints; however, the traditional method of interpreting reproductive impairment may underestimate toxic effects. Cadmium accumulation in liver and ovary of fish increased with increasing Cd exposure concentrations; however, plasma estradiol level remained unaffected. Reproductive capacity in fathead minnows decreased at waterborne Cd concentrations as low as 1xa0μg/L. The findings of our study have important implications for understanding the effects of chronic Cd exposure in metal-impacted feral fish populations.

Determination of Dynamic Metal Complexes and their Diffusion Coefficients in the Presence of Different Humic Substances by Combining Two Analytical Techniques

The combined use of a competing ligand exchange (CLE) method and a diffusive gradient in thin films (DGT) technique in a quasi-labile system provides a better understanding of dynamic metal (Cu and Ni) complexes in the presence of humic substances of different origins. The CLE and DGT techniques provide total labile (dynamic) metal complexes (Cu and Ni) and their dissociation rate constants in environmental systems. DGT was found to estimate lower concentrations of labile metal complexes than CLE. These discrepancies were caused by diffusion controlled metal flux (towards the binding resin gel) in the diffusive gel of DGT. The interactions of Cu and Ni with humic acids are stronger than their interactions with fulvic acid and natural organic matter. Changes in the lability of Ni and Cu complexes (complexed with humic substances of different origins) with the changing analytical detection window indicate that the complexes of these metals were formed with different binding sites with diverse binding energies in the humic substances. The combination of these two techniques was found to be very useful in determining diffusion coefficients of labile metal-humate complexes in quasi-labile systems. The values of diffusion coefficients of labile Ni and Cu complexes determined in this study are in good agreement with limited results from the literature. This finding is novel and can be very useful in further improving our understanding of the metal-humate interactions in natural environments.

Heavy metal bioaccumulation and histopathological alterations in wild Arctic hares (Lepus arcticus) inhabiting a former lead-zinc mine in the Canadian high Arctic: A preliminary study

A preliminary study was undertaken to determine post-mining baseline accumulation of selected trace metals, and histopathological alterations in free-living arctic hares (Lepus arcticus) inhabiting the vicinity of a former lead-zinc mine located on North Baffin Island in the Canadian High Arctic. Trace metal analysis included measurement of As, Cd, Fe, Pb and Zn in tissues, and histopathological assessment comprised of evaluation and scoring the severity of metal-induced hepatic and renal lesions. Metal contents in hepatic and renal tissues from hares from the mine area compared with the reference locations did not differ significantly suggesting that the animals are not uniformly exposed to background levels of metals in the environment. However, relatively higher accumulation pattern of Pb and Cd were noted in liver tissues of hare from the mine area compared to the background area, but did not induce increased lesions. Surface soils near the mine area contained relatively higher levels of trace metals (Zn>Mn>Pb>Cd>As) compared to reference soils, and with soil levels of Cd showing strong correlation with Cd accumulation in kidney tissues. Generally, both case and reference animals showed similar but varying severities of hepatic and renal lesions at the sublethal level, notably vascular congestion, occasional large hepatocyte nuclei, binucleate hepatocytes, yellow-brown pigmentation in the cytoplasm of hepatocytes and clustering of lymphocytes. Only hares with relatively higher accumulation of Pb from the mine area showed evidence of renal edema and hemorrhage of the capsular surface. This study constitutes the first assessment of metal induced histopathological alterations in arctic hares exposed to a historical mining area in the high arctic.

Transport of selenium across the plasma membrane of primary hepatocytes and enterocytes of rainbow trout.

SUMMARY Transport of essential solutes across biological membranes is one of the fundamental characteristics of living cells. Although selenium is an essential micronutrient, little is known about the cellular mechanisms of chemical species-specific selenium transport in fish. We report here the kinetic and pharmacological transport characteristics of selenite and its thiol (glutathione and l-cysteine) derivatives in primary cultures of hepatocytes and isolated enterocytes of rainbow trout. Findings from the current study suggest an apparent low-affinity linear transport system for selenite in both cell types. However, we recorded high-affinity Hill kinetics (Kd=3.61±0.28 μmol l–1) in enterocytes exposed to selenite in the presence of glutathione. The uptake of selenite in the presence of thiols was severalfold higher than uptake of selenite alone (at equimolar concentration) in both hepatocytes and enterocytes. Cellular accumulation of selenium was found to be energy independent. Interestingly, we observed a decrease in selenite transport with increasing pH, whereas selenite uptake increased with increasing pH in the presence glutathione in both cell types. The cellular uptake of selenite demonstrated a pronounced competitive interaction with a structurally similar compound, sulfite. The uptake of selenite as well as its thiol derivatives was found to be sensitive to the anion transport blocker DIDS, irrespective of the cell type. Inorganic mercury (Hg2+) elicited an inhibition of selenite transport in both cell types, but augmented the transport of reduced forms of selenite in hepatocytes. Based on the substrate choice and comparable pharmacological properties, we advocate that multiple anion transport systems are probably involved in the cellular transport of selenite in fish.

The interactive effects of multiple stressors on physiological stress responses and club cell investment in fathead minnows

Anthropogenic activities have dramatically increased over the past decades, with the consequence that many organisms are simultaneously exposed to multiple stressors. Understanding how organisms respond to these stressors is a key focus for scientists from many disciplines. Here we investigated the interactive effects of two stressors, UV radiation (UVR) and cadmium (Cd) exposure on a common freshwater fish, fathead minnow (Pimephales promelas). UVR is known to influence the density of epidermal club cells (ECCs), which are not only a key component of the innate immune system of fishes, but are also the source of chemical alarm cues that serve to warn other fishes of nearby predators. In contrast, Cd impairs the physiological stress response and ability of fish to respond to alarm cues. We used an integrative approach to examine physiological stress response as well as investment in ECCs. Fish exposed to UVR had higher levels of cortisol than non-exposed controls, but Cd reduced cortisol levels substantially for fish exposed to UVR. Fish exposed to UVR, either in the presence or absence of Cd, showed consistent decreases in ECC investment compared to non-exposed controls. Despite differences in ECC number, there was no difference in the potency of alarm cues prepared from the skin of UVR and Cd exposed or non-exposed fish indicating that UVR and Cd exposure combined may have little influence on chemically-mediated predator-prey interactions.

Differential trends in mercury concentrations in double-crested cormorant populations of the Canadian Prairies

Mercury and selenium concentrations were measured in double-crested cormorants (Phalacrocorax auritus), piscivorous fish, and common prey items in five lakes in two ecoregions in Saskatchewan, Canada. Hg and Se concentrations in cormorants were within the natural ranges of birds living in un-impacted sites. Site explained a significant proportion of the variation in total Hg (THg) and methylmercury (MeHg) concentrations in both cormorant breast muscle and livers. Birds nesting on more northern lakes in the Boreal Plain ecoregion (THg rangexa00.11–1.06 and 0.26–9.27xa0μgxa0g−1 wet weight, for breast and liver respectively) had lower THg concentrations compared to those from lakes in the Prairie ecoregion (THg rangexa00.60–4.26xa0μgxa0g−1 ww and 1.59–25.11xa0μgxa0g−1, for breast and liver respectively). Concentrations of MeHg in livers was also lower in birds from northern sites (0.06–1.15xa0μgxa0g−1 ww) compared to those from prairie sites (0.22–4.06xa0μgxa0g−1 ww). We documented a wide range of %MeHg in livers (4.5–52xa0%), indicative of detoxifying MeHg via demethylation to inorganic Hg. Our data suggest that the threshold value where demethylation rates increase substantially appears to be ~10xa0μgxa0g−1 ww MeHg, similar to thresholds in other wildlife. Molar ratios of Hg:Se suggests that some birds from highly saline Reed Lake in the prairie region had insufficient Se available to bind to Hg, thereby removing Se binding as a mitigative strategy for high Hg levels for these birds.

Cadmium transport in isolated enterocytes of freshwater rainbow trout: Interactions with zinc and iron, effects of complexation with cysteine, and an ATPase-coupled efflux

The present study investigated the mechanisms of intestinal cadmium (Cd) uptake and efflux, using isolated enterocytes of freshwater rainbow trout (Oncorhynchus mykiss) as the experimental model. The apical uptake of free Cd(2+) in the enterocytes was a saturable and high-affinity transport process. Both zinc (Zn(2+)) and iron (Fe(2+)) inhibited cellular Cd(2+) uptake through a competitive interaction, suggesting that Cd(2+) enters enterocytes via both Zn(2+) (e.g., ZIP8) and Fe(2+) (e.g., DMT1) transport pathways. Cellular Cd(2+) uptake increased in the presence of HCO(3)(-), which resembled the function of mammalian ZIP8. Cellular Cd(2+) uptake was unaffected by Ca(2+), indicating that Cd(2+) does not compete with Ca(2+) for apical uptake. Interestingly, Cd uptake was influenced by the presence of l-cysteine, and under the exposure condition where Cd(Cys)(+) was the predominant Cd species, cellular Cd uptake rate increased with the increased concentration of Cd(Cys)(+). The kinetic analysis indicated that the uptake of Cd(Cys)(+) occurs through a low capacity transport mechanism relative to that of free Cd(2+). In addition, Cd efflux from the enterocytes decreased in the presence of an ATPase inhibitor (orthovanadate), suggesting the existence of an ATPase-coupled extrusion process. Overall, our findings provide new mechanistic insights into the intestinal Cd transport in freshwater fish.

Influence of elevated alkalinity and natural organic matter (NOM) on tissue-specific metal accumulation and reproductive performance in fathead minnows during chronic, multi-trophic exposures to a metal mine effluent

Metal bioavailability in aquatic organisms is known to be influenced by various water chemistry parameters. The present study examined the influence of alkalinity and natural organic matter (NOM) on tissue-specific metal accumulation and reproductive performance of fathead minnows (Pimephales promelas) during environmentally relevant chronic exposures to a metal mine effluent (MME). Sodium bicarbonate (NaHCO3) or NOM (as commercial humic acid) were added to a Canadian MME [45 percent process water effluent (PWE)] in order to evaluate whether increases in alkalinity (3-4 fold) or NOM (~1.5-3mg/L dissolved organic carbon) would reduce metal accumulation and mitigate reproductive toxicity in fathead minnows during a 21-day multi-trophic exposure. Eleven metals (barium, boron, cobalt, copper, lithium, manganese, molybdenum, nickel, rubidium, selenium, and strontium) were elevated in the 45 percent PWE relative to the reference water. Exposure to the unmodified 45 percent PWE resulted in a decrease of fathead minnow egg production (~300 fewer eggs/pair) relative to the unmodified reference water, over the 21-day exposure period. Water chemistry modifications produced a modest decrease in free ion activity of some metals (as shown by MINTEQ, Version 3) in the 45 percent PWE exposure water, but did not alter the metal burden in the treatment-matched larval Chironomus dilutus (the food source of fish during exposure). The tissue-specific metal accumulation increased in fish exposed to the 45 percent PWE relative to the reference water, irrespective of water chemistry modifications, and the tissue metal concentrations were found to be similar between fish in the unmodified and modified 45 percent PWE (higher alkalinity or NOM) treatments. Interestingly however, increased alkalinity and NOM markedly improved fish egg production both in the reference water (~500 and ~590 additional eggs/pair, respectively) and 45 percent PWE treatments (~570 and ~260 additional eggs/pair, respectively), although fecundity over 21 day exposure consistently remained lower in the 45 percent PWE treatment groups relative to the treatment-matched reference groups. Collectively, these findings suggest that metal accumulation caused by chronic 45 percent PWE exposure cannot solely explain the reproductive toxicity in fish, and decrease in food availability (decrease in C. dilutus abundance in 45 percent PWE exposures) might have played a role. In addition, it appears that NaHCO3 or humic acid mitigated reproductive toxicity in fish exposed to 45 percent PWE by their direct beneficial effects on the physiological status of fish.