Why Does Cysteine Enhance Metal Uptake by Phytoplankton in Seawater but Not in Freshwater?

Low-molecular-weight weak ligands such as cysteine have been shown to enhance metal uptake by marine phytoplankton in the presence of strong ligands, but the effect is not observed in freshwater. We hypothesized that these contrasting results might be caused by local cysteine degradation and a Ca effect on metal-ligand exchange kinetics in the boundary layer surrounding the algal cells; newly liberated free metal ions cannot be immediately complexed in seawater by Ca-bound strong ligands but can be rapidly complexed by free ligands at low-Ca levels. The present results consistently support this hypothesis. At constant bulk Cd2+ concentrations, buffered by strong ligands: (1) at 50 mM Ca, cysteine addition significantly enhanced Cd uptake in high-Ca preacclimated euryhaline Chlamydomonas reinhardtii (cultured with cysteine as a nitrogen source to enhance local Cd2+ liberation via cysteine degradation); (2) at 0.07 mM Ca, this enhancement was not observed in the algae; (3) at 50 mM Ca, the enhancement disappeared when C. reinhardtii were cultured with ammonium (to inhibit cysteine degradation and local Cd2+ liberation); (4) cysteine addition did not enhance Cd uptake by cysteine-cultured marine Thalassiosira weissflogii when the concentration of immediately reacting strong ligands was sufficient to complex local Cd2+ liberation.

Uptake and subcellular distribution of aluminum in a marine diatom.

Aluminum (Al) is widespread in the environment including the ocean. The effects of Al on marine organisms have attracted more and more attention in recent years. However, the mechanisms of uptake of Al by marine organisms and the subcellular distribution of Al once assimilated are unknown. Here we report the uptake and subcellular distribution of Al in a marine diatom Thalassiosira weissflogii. Short-term (< 120 min) uptake experiments showed that the Al uptake rate by the diatom was 0.033 ±â€¯0.013 fmol-1 cell-1 min-1 (internalization flux normalized to the exposure Al concentration of 2 µM = 0.034 ±â€¯0.013 nmol m-2 min-1 nM-1). Subcellular fractionation experiments showed that the internalized Al was partitioned to subcellular components in the following order: granules (69 ±â€¯5%) > debris (17 ±â€¯4%) > organelles (12 ±â€¯2%) > heat-stable peptides (HSP) (~2%) > heat-denaturable proteins (HDP) (< 1%), indicating that the majority of intracellular Al was detoxified and stored in inorganic forms. The subcellular distribution of Al in the diatom is different from that of Al in freshwater green algae, in which most of the internalized Al is partitioned to organelles. We also evaluated an artificial seawater-based EDTA rinse solution to remove Al adsorbed on the diatom cell surface. Overall, our study provides new information to understand the mechanisms of uptake of Al by marine diatoms, and the mechanisms responsible for the biological effects (both toxic and beneficial) of Al on the growth of marine phytoplankton, especially diatoms.

Chemical Conditions in the Boundary Layer Surrounding Phytoplankton Cells Modify Cadmium Bioavailability.

In this study we tested the hypothesis that metal uptake by unicellular algae may be affected by changes in metal speciation in the boundary layer surrounding the algal cells. The freshwater alga Chlamydomonas reinhardtii was preacclimated to different N nutrition regimes; changes in N nutrition are known to change the nature of extracellular metabolites (e.g., reactive oxygen species "ROS", and OH-) and thus boundary layer chemical conditions. Specifically, at a constant bulk free Cd2+ concentration, Cd uptake by N-starved algae in cysteine-buffered solution was significantly higher than that in NTA-buffered solution. This enhancement was likely due to an increase of the free Cd2+ concentration in the boundary layer, resulting from localized cysteine oxidation by ROS released from these algae. On the other hand, Cd uptake was markedly lower when the free Cd2+ concentration near cell surface decreased as a result of an increase in the boundary layer pH of nitrate-acclimated algae or enhanced localized metal complexation. The results imply that redox, acid-base and metal complexation processes in the boundary layer differ from those in bulk water, even under chemically stable bulk conditions, and the boundary layer effect may well be of significance to phytoplankton acquisition of other trace metals.

Binding of trace elements (Ag, Cd, Co, Cu, Ni, and Tl) to cytosolic biomolecules in livers of juvenile yellow perch (Perca flavescens) collected from lakes representing metal contamination gradients.

Biomolecules involved in handling cytosolic metals in the liver of the yellow perch (Perca flavescens) were characterized in juvenile fish collected from 4 lakes constituting metal contamination gradients. Using size-exclusion liquid chromatography coupled to an inductively coupled mass spectrometer, we determined metal distributions among ligands of different molecular weights in the cytosol, before and after a heat denaturation step designed to isolate metallothionein-like peptides and proteins. Silver, Cd, and Cu found in the heat-stable protein supernatants were indeed largely present as metallothionein-like peptide complexes; but Co, Ni, and Tl, also present in the heat-stable protein supernatants, did not coelute with metallothionein-like peptides and proteins. This difference in metal partitioning is consistent with the known preference of "soft" metals such as Ag, Cd, and Cu(I) for thiolated ligands and the contrasting tendency of Co and Ni to bind to ligands with oxygen and nitrogen as donor atoms. Metal handling in the whole cytosol also reflected these differences in metal-binding behavior. For Cd and Cu, the importance of the molecular weight pool that includes metallothionein-like peptides and proteins increased relative to the other pools as the total cytosolic metal concentration ([M]cytosol ) increased, consistent with a concentration-dependent detoxification response. In contrast, for Ni and Tl the increase in [M]cytosol was accompanied by a marked increase in the high-molecular weight (670-33 kDa) pool, suggesting that hepatic Ni and Tl are not effectively detoxified. Overall, the results suggest that metal detoxification is less effective for Ni, Tl, and Co than for Ag, Cd, and Cu. Environ Toxicol Chem 2018;37:576-586. © 2017 SETAC.

Hepatic oxidative stress and metal subcellular partitioning are affected by selenium exposure in wild yellow perch (Perca flavescens).

Yellow perch (Perca flavescens) collected from 11 lakes in the Canadian mining regions of Sudbury (Ontario) and Rouyn-Noranda (Quebec) display wide ranges in the concentrations of cadmium (Cd), nickel (Ni), selenium (Se), and thallium (Tl) in their livers. To determine if these trace elements, as well as copper (Cu) and zinc (Zn), are causing oxidative stress in these fish, we measured three biochemical indicators (glutathione (GSH), glutathione disulfide (GSSG) and thiobarbituric acid-reactive substances (TBARS)) in their livers. We observed that 44% of the yellow perch that we collected were at risk of cellular oxidative stress and lipid peroxidation. Considering all fish from all lakes, higher liver Se concentrations were coincident with both lower proportions of GSSG compared to GSH and lower concentrations of TBARS, suggesting that the essential trace-element Se acts as an antioxidant. Furthermore, fish suffering oxidative stress had higher proportions of Cd, Cu and Zn in potentially sensitive subcellular fractions (organelles and heat-denatured proteins) than did fish not suffering from stress. This result suggests that reactive oxygen species may oxidize metal-binding proteins and thereby reduce the capacity of fish to safely bind trace metals. High Cd concentrations in metal-sensitive subcellular fractions likely further exacerbate the negative effects of lower Se exposure.

Transcriptional and biochemical markers in transplanted Perca flavescens to characterize cadmium- and copper-induced oxidative stress in the field.

Despite recent progress achieved in elucidating the mechanisms underlying local adaptation to pollution, little is known about the evolutionary change that may be occurring at the molecular level. The goal of this study was to examine patterns of gene transcription and biochemical responses induced by metal accumulation in clean yellow perch (Perca flavescens) and metal depuration in contaminated fish in a mining and smelting region of Canada. Fish were collected from a reference lake (lake Opasatica) and a Cd, Cu and Zn contaminated lake (lake Dufault) located in the Rouyn-Noranda region (Qc, Canada) and caged for one or four weeks in their own lake or transplanted in the other lake. Free-ranging fish from the same lakes were also collected. Kidney Cd and Cu concentrations in clean fish caged in the contaminated lake increased with the time of exposure, but metal depuration did not occur in contaminated fish caged in the clean lake. After 4 weeks, the major retinoid metabolites analysed, the percentage of free dehydroretinol (dROH) and the retinol dehydrogenase-2 (rdh-2) transcription level in liver decreased in clean fish transplanted into the metal-contaminated lake, suggesting that metal exposure negatively impacted retinoid metabolism. However, we observed an increase in almost all of the retinoid parameters analysed in fish from the metal-impacted lake caged in the same lake, which we interpret as an adaptation response to higher ambient metal concentration. In support of this hypothesis, liver transcription levels of microsomal glutathione-S-transferase-3 (mgst-3) and glucose-6-phosphate dehydrogenase (g6pdh) were enhanced in clean fish transplanted into the metal-contaminated lake and this up-regulation was accompanied by an increase in the activities of corresponding enzymes, involved in antioxidant response. However, although in the same fish the transcription level of Cu/Zn superoxide dismutase (Cu/Zn sod) was also increased, this did not lead to a change in the activity of the SOD enzyme, suggesting that this upregulation was aimed at maintaining SOD-related antioxidant capacities. In contrast, the transcription level of the cat gene, which did not change in contaminated fish, did not compensate for the decrease of CAT activity. After 4 weeks of exposure, some plastic responses of the clean fish were observed when they were transplanted in the metal-contaminated lake. However, probably as a consequence of the prior 80 years of exposure to metals, the contaminated population showed a limited plastic response in the expression of the majority of the candidate genes tested, when they were transplanted in the reference lake. The overall findings of this field investigation highlight how yellow perch molecularly and biochemically responded to a sudden or relatively long-term exposure (4 weeks) to a cocktail of metals.

Cadmium accumulation and toxicity in the unicellular alga Pseudokirchneriella subcapitata: Influence of metal-binding exudates and exposure time.

Predicting metal availability and toxicity for chronic (several hours or days) metal exposure scenarios, even for unicellular algae, is a major challenge to existing toxicity models. This is because several factors affecting metal uptake and toxicity, such as the release of metal-binding exudates, changes in the kinetics of metal uptake and toxicity over time, and algal physiological acclimation to internalized metals, are still poorly understood. The present study assessed the influence of these factors on Cd uptake and toxicity in laboratory batch cultures of the freshwater alga Pseudokirchneriella subcapitata. To do so, changes in the free Cd(2+) concentrations caused by the release of metal-binding algal exudates were monitored, (109)Cd accumulation in algal cells was measured, and Cd-induced inhibition of algal growth as a function of exposure time (from 12 h to 96 h) was followed. Results indicate that metal-binding exudates may decrease the proportion of the free Cd(2+) ion in solution up to 2-fold, a decrease that affects Cd uptake and toxicity. Pseudokirchneriella subcapitata has the capacity to decrease net Cd uptake rate on short time scales (<24 h), but this reduction in the Cd uptake rate disappeared after 24 h, and Cd toxicity occurred at relatively high Cd concentrations in solution. These data illustrate some of the pitfalls of standard algal toxicity assays, which were designed for acute exposures, and suggest how robust chronic bioassays might be developed.

Uptake and subcellular distributions of cadmium and selenium in transplanted aquatic insect larvae.

We transplanted larvae of the phantom midge Chaoborus punctipennis from a lake having lower concentrations of Cd and Se (Lake Dasserat) to a more contaminated lake (Lake Dufault) located near a metal smelter in Rouyn-Noranda, Quebec. Transplanted individuals were held in mesh mesocosms for up to 16 days where they were fed with indigenous contaminated zooplankton. Larval Cd and Se burdens increased over time, and came to equal those measured in indigenous C. punctipennis from contaminated Lake Dufault. Larval Se burdens increased steadily, whereas those of Cd showed an initial lag phase that we explain by a change in the efficiency with which this insect assimilated Cd from its prey. We measured Cd and Se in subcellular fractions and found that larvae sequestered the majority (60%) of the incoming Cd in a detoxified fraction containing metal-binding proteins, whereas a minority of this nonessential metal was in sensitive fractions (20%). In contrast, a much higher proportion of the essential element Se (40%) was apportioned to metabolically active sensitive fractions. Larvae took up equimolar quantities of these elements over the course of the experiment. Likewise, Cd and Se concentrations in wild larvae were equimolar, which suggests that they are exposed to equimolar bioavailable concentrations of these elements in our study lakes.

Waterborne cadmium and nickel impact oxidative stress responses and retinoid metabolism in yellow perch.

In this experiment, we studied the transcriptional and functional (enzymatic) responses of yellow perch (Perca flavescens) to metal stress, with a focus on oxidative stress and vitamin A metabolism. Juvenile yellow perch were exposed to two environmentally relevant concentrations of waterborne cadmium (Cd) and nickel (Ni) for a period of 6 weeks. Kidney Cd and Ni bioaccumulation significantly increased with increasing metal exposure. The major retinoid metabolites analyzed in liver and muscle decreased with metal exposure except at high Cd exposure where no variation was reported in liver. A decrease in free plasma dehydroretinol was also observed with metal exposure. In the liver of Cd-exposed fish, both epidermal retinol dehydrogenase 2 transcription level and corresponding enzyme activities retinyl ester hydrolase and lecithin dehydroretinyl acyl transferase increased. In contrast, muscle epidermal retinol dehydrogenase 2 transcription level decreased with Cd exposure. Among antioxidant defences, liver transcription levels of catalase, microsomal glutathione-S-transferase-3 and glucose-6-phosphate dehydrogenase were generally enhanced in Cd-exposed fish and this up-regulation was accompanied by an increase in the activities of corresponding enzymes, except for microsomal glutathione-S-transferase. No consistent pattern in antioxidant defence responses was observed between molecular and biochemical response when fish were exposed to Ni, suggesting a non-synchronous response of antioxidant defence in fish exposed to waterborne Ni. There was a general lack of consistency between muscle transcription level and enzyme activities analyzed. The overall findings from this investigation highlight the usefulness of transcriptional and biochemical endpoints in the identification of oxidative stress and vitamin A metabolism impairment biomarkers and the potential use of multi-level biological approaches when assessing environmental risk in fish.

Predicting cadmium accumulation and toxicity in a green alga in the presence of varying essential element concentrations using a biotic ligand model.

This study refines the Biotic Ligand Model (BLM) approach by integrating the modulating effects of various essential elements on cadmium (Cd) uptake kinetics in the freshwater alga Chlamydomonas reinhardtii. The algae were first acclimated to a low (LM) or high trace metal (HM) medium as well as to low or high free Cd(2+) and Co(2+) concentrations. The short-term Cd transport capacity and affinity were then quantified in exposure media in which essential trace metals and calcium concentrations were manipulated. The results show that after acclimation to the LM medium, exposure to high free Ca(2+) decreases the capacity of the Cd transport system. Also, acclimation to high (10(-9) M free Co(2+)) or low (10(-11) M free Co(2+)) did not significantly affect Cd uptake rates. When all essential trace metals were simultaneously increased in the acclimation (and exposure) medium, the capacity of the transport system decreased by ∼ 60%, a decrease close to that due to high [Zn(2+)] alone, suggesting that Zn is the main trace metal modulator of the Cd transporter capacity. Changes in Cd toxicity (growth inhibition) in the presence of different essential trace metal concentrations were strongly related to the steady-state concentration of intracellular cadmium, regardless of the cell's nutritional state. Our BLM incorporating the physiological effects of Ca(2+) and other trace metals predicts steady-state Cd accumulation in the presence of varying concentrations of essential elements at 7 nM free Cd(2+), but predictions over a wide range of free [Cd(2+)] proved to be more difficult.

How does exposure to nickel and cadmium affect the transcriptome of yellow perch (Perca flavescens)--results from a 1000 candidate-gene microarray.

The molecular mechanisms underlying nickel (Ni) and cadmium (Cd) toxicity and their specific effects on fish are poorly understood. Documenting gene transcription profiles offers a powerful approach toward identifying the molecular mechanisms affected by these metals and to discover biomarkers of their toxicity. However, confounding environmental factors can complicate the interpretation of the results and the detection of biomarkers for fish captured in their natural environment. In the present study, a 1000 candidate-gene microarray, developed from a previous RNA-seq study on a subset of individual fish from contrasting level of metal contamination, was used to investigate the transcriptional response to metal (Ni and Cd) and non metal (temperature, oxygen, and diet) stressors in yellow perch (Perca flavescens). Specifically, we aimed at (1) identifying transcriptional signatures specific to Ni and Cd exposure, (2) investigating the mechanisms of their toxicity, and (3) developing a predictive tool to identify the sublethal effects of Ni and Cd contaminants in fish sampled from natural environments. A total of 475 genes displayed significantly different transcription levels when temperature varied while 287 and 176 genes were differentially transcribed at different concentrations of Ni and Cd, respectively. These metals were found to mainly affect the transcription level of genes involved in iron metabolism, transcriptional and translational processes, vitamin metabolism, blood coagulation, and calcium transport. In addition, a linear discriminant analysis (LDA) made using gene transcription levels yielded 94% correctly reassigned samples regarding their level of metal contamination, which indicates the potential of the microarray to detect perch response to Cd or Ni effects.

Extending the biotic ligand model to account for positive and negative feedback interactions between cadmium and zinc in a freshwater alga.

Low concentrations of essential trace metals such as zinc (Zn) were recently shown to strongly modulate cadmium (Cd) uptake in the freshwater alga Chlamydomonas reinhardtii. Here we studied the mechanisms of Cd and Zn acquisition by this alga, using metal uptake kinetics experiments. Cadmium uptake rates fitted a three transport site model characterized by the affinity constants K(Cd­1)(Cd) = 10(5.0), K(Cd­2)(Cd) = 10(7.6), and K(Cd­3)(Cd) = 10(8.8). Similar uptake kinetics were obtained for Zn with K(Zn­1)(Zn) = 10(5.0), K(Zn­2)(Zn) = 10(7.4), and K(Zn­3)(Zn) > 10(9). Competitive binding experiments suggest that Zn and Cd share the same three transport systems. The capacities of the transport systems were modulated by as much as 10-fold following preacclimation to high or low Zn(2+) and Cd(2+) concentrations. We conclude that the strong protective effect of Zn on Cd accumulation is mainly due to the reduction of the maximal uptake rate of the high-affinity Zn­2 (or Cd­2) transport system. A biotic ligand model was developed to incorporate the effects of both chemical speciation and physiological regulation of Cd transport systems. The model successfully predicts the experimentally measured steady-state Cd content of C. reinhardtii in the presence of low or high [Zn(2+)].

Influence of essential elements on cadmium uptake and toxicity in a unicellular green alga: the protective effect of trace zinc and cobalt concentrations.

Within the biotic ligand model (BLM) construct, major cations are considered to be simple competitors for metal binding to uptake sites and may offer some protection against metal-induced toxicity, but the influence of essential trace elements and cell preconditioning to different micronutrient concentrations on metal uptake and toxicity is considered negligible. To test these underlying assumptions, we monitored Cd uptake and toxicity in a green alga (Chlamydomonas reinhardtii) after long-term exposures (60 h) to a range of environmentally realistic free Zn(2+) , Co(2+) , Fe(3+) , Mn(2+) , Ca(2+) , and Cu(2+) concentrations buffered with nitrilotriacetic acid. A 200-fold increase in free [Mn(2+) ] as well as a 100-fold increase in free [Fe(3+) ] did not affect Cd uptake or toxicity, whereas a 50-fold increase in free [Ca(2+) ] effectively offered some protection, as predicted by the BLM. However, a 10-fold increase in free [Cu(2+) ] significantly enhanced Cd toxicity by a factor of approximately 2, whereas a 100-fold increase in free [Zn(2+) ] and [Co(2+) ] from 10(-11) to 10(-9) M significantly decreased Cd uptake and toxicity by more than twofold. These effects did not change with prior algal acclimation to different essential micronutrient concentrations. Low essential trace metal concentrations may strongly affect the uptake and toxicity of Cd in freshwater algae and should be taken into consideration in future developments of the BLM.

UPTAKE OF LIPOPHILIC CADMIUM COMPLEXES BY THREE GREEN ALGAE: INFLUENCE OF HUMIC ACID AND ITS pH DEPENDENCE(1).

Cadmium forms neutral, lipophilic CdL2 (0) complexes with diethyldithiocarbamate (L = DDC) and with ethylxanthate (L = XANT). In a synthetic solution and in the absence of natural dissolved organic matter (DOM), for a given total Cd concentration, uptake of these complexes by unicellular algae is much faster than the uptake of the free Cd(2+) cation. The objective of the present study was to determine how this enhanced uptake of the lipophilic CdL2 (0) complexes was affected by the presence of natural DOM (Suwannee River humic acid, SRHA). Experiments were performed with Cd(DDC)2 (0) and Cd(XANT)2 (0) at two pH values (7.0 and 5.5) and with the three chlorophytes [Chlamydomonas reinhardtii P. A. Dang., Pseudokirchneriella subcapitata (Korshikov) Hindák, Chlorella fusca var. vacuolata Shihira et R. W. Krauss]. Short-term uptake (30-40 min) of the CdL2 (0) complexes was followed in the absence and presence of SRHA (6.5 mg C · L(-1) ). Acidification from pH 7.0 to 5.5 decreased CdL2 (0) uptake by the three algae, in the presence or absence of humic acid (HA). The dominant effect of the HA was to decrease Cd uptake, due to its interaction with the CdL2 (0) complexes in solution. However, if uptake of the free CdL2 (0) complexes was compared in the presence and absence of HA, in four of eight cases initial uptake rate constants (ki ) were significantly higher (P < 0.05) in the presence of the HA, suggesting the operation of an interfacial effect of the HA at the algal cell membrane, favoring uptake of CdL2 (0) . Overall, the experimental results suggest that neutral metal complexes will be less bioavailable in natural waters than they are in synthetic laboratory media in the absence of natural DOM.

Subcellular partitioning of cadmium in the freshwater bivalve, Pyganodon grandis, after separate short-term exposures to waterborne or diet-borne metal.

The dynamics of cadmium uptake and subcellular partitioning were studied in laboratory experiments conducted on Pyganodon grandis, a freshwater unionid bivalve that shows promise as a biomonitor for metal pollution. Bivalves were collected from an uncontaminated lake, allowed to acclimate to laboratory conditions (≥25 days), and then either exposed to a low, environmentally relevant, concentration of dissolved Cd (5nM; 6, 12 and 24h), or fed Cd-contaminated algae (∼70nmol Cdg⁻¹ dry weight; 4×4h). In this latter case, the bivalves were allowed to depurate for up to 8 days after the end of the feeding phase. As anticipated, the gills were the main target organ during the aqueous Cd exposure whereas the intestine was the initial site of Cd accumulation during the dietary exposure; during the subsequent depuration period, the dietary Cd accumulated in both the digestive gland and in the gills. For the gills, the distribution of Cd among the subcellular fractions (i.e., granules>heat-denatured proteins (HDP)∼heat-stable proteins (HSP)>mitochondria∼lysosomes+microsomes) was insensitive to the exposure route; both waterborne and diet-borne Cd ended up largely bound to the granule fraction. The subcellular distribution of Cd in the digestive gland differed markedly from that in the gills (HDP>HSP∼granules∼mitochondria>lysosomes+microsomes), but as in the case of the gills, this distribution was relatively insensitive to the exposure route. For both the gills and the digestive gland, the subcellular distributions of Cd differed from those observed in native bivalves that are chronically exposed to Cd in the field - in the short-term experimental exposures of P. grandis, metal detoxification was less effective than in chronically exposed native bivalves.

Responses of two sentinel species (Hexagenia limbata--mayfly; Pyganodon grandis--bivalve) along spatial cadmium gradients in lakes and rivers in northwestern Québec.

Specimens of the mayfly larva Hexagenia limbata and of the floater mussel Pyganodon grandis were sampled in rivers and lakes contaminated by trace metals in the Abitibi-James Bay region in northwestern Québec. Water samples were collected at each sampling site with in situ diffusion samplers and analyzed for major cations, anions and trace metals (Cd, Cu, Mn, Zn). Surficial sediment samples were also collected at each site and analyzed for Cd, Cu and Zn. In response to Cd contamination at river and lake sites, both sentinel organisms accumulated the metal and synthesized metallothionein (MT), a metal-binding protein synthesized by organisms as a defence mechanism against excess metals in the surrounding media. At the river sites, H. limbata unexpectedly maintained much higher concentrations of MT per unit of accumulated Cd than at the lake sites; this difference between lentic and lotic environments may reflect the response of the species to the more stressful hydrodynamic conditions that prevail in a river. The accumulation of Cd in the mayflies at lake and river sites decreased as a function of the ambient manganese concentration. We hypothesize that dissolved Mn protects against Cd bioaccumulation in H. limbata. The present results support the contention that one cannot extrapolate conclusions drawn from the use of a single sentinel species to a larger set of freshwater invertebrates--both the mayfly and the bivalve are promising biomonitors.

Cadmium detoxification strategies in two phytoplankton species: metal binding by newly synthesized thiolated peptides and metal sequestration in granules.

The aim of this study was to evaluate whether intracellular detoxification mechanisms could explain, at least partially, the different sensitivity to Cd of two freshwater green algae, Chlamydomonas reinhardtii and Pseudokirchneriella subcapitata. Subcellular Cd distribution and the synthesis of metal-binding thiolated peptides were thus examined in both algae exposed to a range of free [Cd(2+)] from 0.7 to 253 nM. Cadmium partitioning among five subcellular fractions (cellular debris, granules, organelles, heat-denaturable proteins - HDP, and heat-stable proteins - HSP) was determined after differential centrifugation of algal homogenates. Thiolated-peptides, phytochelatins (PC(n)) and precursors, were analyzed by HPLC with pre-column monobromobimane derivatization. Cadmium accumulation per cell was 2-4 times greater for C. reinhardtii than for P. subcapitata, yet C. reinhardtii was more resistant to Cd with an EC(50) of 273 nM Cd(2+) [244-333 nM Cd(2+) CI(95%)]) compared to 127 nM Cd(2+) [111-143 nM Cd(2+) CI(95%)] for P. subcapitata. Although [Cd] generally increased in the organelle fractions when free [Cd(2+)] increased in the experimental media, their relative contributions to the total Cd cellular content decreased, suggesting that partial protection of some metal sensitive sites was achieved by the initiation of cellular detoxification mechanisms. An increase in the proportion of Cd in the granules fraction was observed for C. reinhardtii between 6 and 15 nM Cd(2+) (i.e., at [Cd(2+)]

pH modulates transport rates of manganese and cadmium in the green alga Chlamydomonas reinhardtii through non-competitive interactions: implications for an algal BLM.

The influence of pH on short-term uptake of manganese and cadmium by the green alga Chlamydomonas reinhardtii was studied to better understand the nature of proton interactions with metal membrane transporters. Manganese and cadmium internalization fluxes (J(int)) were measured over a wide range of free metal ion concentrations from 1 x 10(-10) to 4 x 10(-4)M at several pH values (Mn: 5.0, 6.5 and 8.0; Cd: 5.0 and 6.5). For both metals, first-order biological internalization kinetics were observed but the maximum transport flux (J(max)) decreased when pH decreased, in contradiction with the Biotic Ligand Model (BLM). This result suggested a non-competitive inhibition of metal uptake by the H(+)-ion. A Michaelis-Menten type inhibition model considering proton and calcium competition was tested. The metal biotic ligand stability constants and the stability constants for competitive binding of Ca(2+) and H(+) with the metal transporters were calculated: for manganese, K(Mn)=10(4.20) and K(Ca)=10(3.71); for cadmium, K(Cd)=10(4.19) and K(Ca)=10(4.76); for both metal transport systems, K(H) was not a significant parameter. Furthermore, metal uptake was not significantly influenced by the pH of the antecedent growth medium, suggesting that increases in metal fluxes as the pH is raised are caused by conformational changes of the surface transport proteins rather than by the synthesis of additional transport sites. Our results demonstrate that the BLM in its present state does not properly describe the true influence of pH on manganese and cadmium uptake by algae and that a non-competitive inhibition component must be integrated.

Linking changes in subcellular cadmium distribution to growth and mortality rates in transplanted freshwater bivalves (Pyganodon grandis).

Relationships between Cd accumulation and subcellular distribution, and growth and mortality rates were examined in the freshwater bivalve Pyganodon grandis in a transplant experiment. Organisms were transferred from a clean lacustrine site to four lakes situated along a Cd concentration gradient in the mining region of Rouyn-Noranda. The bivalves were maintained in open enclosures placed in the bottom sediments of the littoral zone of all five lakes for 400 days. At the end of the experiment, metallothionein (MT) was measured in the bivalve gills with a Hg-saturation assay and Cd partitioning among the various cytosolic protein pools was determined by size-exclusion chromatography. Marked differences were observed among the five sites: the range in calculated free-cadmium ion concentrations in water overlying the sediments was 35-fold whereas Cd concentrations in the gill cytosol of the transplanted bivalves varied three-fold. In the transplanted bivalves, the distribution of gill Cd among the various cytosolic complexes also varied significantly among sites. For bivalves transplanted to the three most contaminated sites, Cd concentrations in the high molecular weight pool (HMW>25 kDa) were significantly higher than the baseline levels determined from bivalves caged at the reference site; a similar trend was seen for Cd concentrations in the metallothionein pool (Cd-MT). For bivalves transferred to two of the high contamination sites, proportionately less of the gill cytosolic Cd was sequestered (i.e. detoxified) by MT-like proteins. Reductions in survival were also observed at these two sites, and these elevated mortalities, in turn, were consistent with the absence of indigenous bivalve populations at these sites. This result is compatible with our recent work on P. grandis populations living in lakes of the Rouyn-Noranda area, in which we demonstrated that excessive accumulation of Cd in the HMW pool of the gill cytosol of the individual mollusks could be related to the impairment of population health status.

Sub-cellular partitioning of cadmium, copper, nickel and zinc in indigenous yellow perch (Perca flavescens) sampled along a polymetallic gradient.

Sub-cellular metal distributions were studied in indigenous yellow perch (Perca flavescens) collected from eight lakes located along a cadmium (Cd), copper (Cu), nickel (Ni) and zinc (Zn) concentration gradient. Ambient dissolved metal concentrations were measured to evaluate exposure and total hepatic metal concentrations were determined as a measure of metal bioaccumulation. Metal partitioning among potentially metal-sensitive fractions (cytosolic enzymes, organelles) and detoxified metal fractions (metallothionein) was determined after differential centrifugation of fish liver homogenates. Major proportions of hepatic Cd and Cu were found in the heat-stable cytosolic peptides and proteins fraction (HSP), a fraction including metallothioneins, whereas the potentially metal-sensitive heat-denaturable proteins fraction (HDP) was the largest contributor to the total Ni and Zn burdens. The concentrations of Cd, Cu and Ni (but not Zn) in each sub-cellular fraction increased along the metal contamination gradient, but the relative contributions of each fraction to the total burden of each of these metals remained generally constant. For these chronically exposed fish there was no threshold exposure concentration below which binding of Cd or Ni to the heat-denaturable protein fraction did not occur. The presence of Cd and Ni in the HDP fraction, even for low chronic exposure concentrations, suggests that metal detoxification was imperfect, i.e. that P. flavescens was subject to some metal-related stress even under these conditions.