Sublethal Cd Exposure Research Articles

High-level dietary cadmium exposure is associated with global DNA hypermethylation in the gastropod hepatopancreas.

5-methylcytosine (5mC) is a key epigenetic mark which influences gene expression and phenotype. In vertebrates, this epigenetic mark is sensitive to Cd exposure, but there is no information linking such an event with changes in global 5mC levels in terrestrial gastropods despite their importance as excellentecotoxicological bioindicators of metal contamination. Therefore, we first evaluated total 5mC content in DNA of the hepatopancreas of adult Cantareus aspersus with the aim to determine whether this epigenetic mark is responsive to Cd exposure. The experiment was conducted under laboratory conditions and involved a continuous exposure, multiple dose- and time-point (14, 28, and 56 days) study design. Hepatopancreas cadmium levels were measured using Flame Atomic Absorption Spectrometry and the percentage of 5-mC in samples using an ELISA-based colorimetric assay. Snail death rates were also assessed. Our results, for the first time, reveal the presence of 5mC in C. aspersus and provide evidence for Cd-induced changes in global 5mC levels in DNA of gastropods and mollusks. Although less sensitive than tissue accumulation, DNA methylation levels responded in a dose- and time-dependent manner to dietary cadmium, with exposure dose having a much stronger effect than exposure duration. An obvious trend of increasing 5mC levels was observed starting at 28 days of exposure to the second highest dose and this trend persisted at the two highest treatments for close to one month, when the experiment was terminated after 56 days. Moreover, a strong association was identified between Cd concentrations in the hepatopancreas and DNA methylation levels in this organ. These data indicate an overall trend towards DNA hypermethylation with elevated Cd exposure. No consistent lethal effect was observed, irrespective of time point and Cd-dosage. Overall, our findings suggest that the total 5mC content in DNA of the hepatopancreas of land snails is responsive to sublethal Cd exposure and give new insights into invertebrate environmental epigenetics.

Metabolic responses of Arabidopsis thaliana roots and leaves to sublethal cadmium exposure are differentially influenced by ALTERNATIVE OXIDASE1a

The mitochondrial alternative oxidase (AOX) is suggested to modulate metabolic homeostasis and is activated at transcript and protein level in cadmium (Cd)-exposed Arabidopsis thaliana plants. In this study, the importance of AOX in the metabolic response to Cd stress was investigated. Primary metabolites were determined in roots and leaves of wild-type plants and aox1a knockout mutants exposed to sublethal Cd concentrations for 24 and 72h. Additional parameters (gene expression, enzyme capacity) were included to support the results at the metabolic level. Whereas sugar and organic acid levels initially dropped in roots, the opposite was observed in leaves of both genotypes exposed to Cd. Regarding genotype-related differences in the leaves, our results strengthen the previously proposed link between AOX1a and ethylene. Furthermore, respiratory flux dependent on amino acid degradation could be related to AOX1a function in leaves of Cd-exposed plants. In roots, a relation between AOX1a and antioxidative defence – in particular glutathione (GSH) – is demonstrated during Cd exposure. In conclusion, A. thaliana plants show a tissue-specific response to sublethal Cd exposure, with a differential role for AOX1a in roots and leaves.

The effects of chronic cadmium exposure on repeat swimming performance and anaerobic metabolism in brown trout (Salmo trutta) and lake whitefish (Coregonus clupeaformis)

This study investigates the effect of chronic Cd exposure on the ability to perform repeat swim challenges in brown trout (Salmo trutta) and lake whitefish (Coregonus clupeaformis). Fish were exposed to waterborne Cd (18nM) in moderately hard water (120mgL−1 CaCO3) for 30 days. This level of exposure has been shown to cause sublethal physiological disruption and acclimation responses but no impairment of sustained swimming capacity (Ucrit) in single swim challenges. Swim trials were done over the course of the exposure and each one consisted of an initial swim to 85% of the Ucrit of control fish, a 30min recovery period and finally a second swim challenge to determine Ucrit. Plasma and tissue samples were collected before and after each of the swim periods. As expected from previous studies, Cd exposure resulted in significant accumulation of Cd in gills, liver and kidney but not in white muscle. Exposure also induced a loss of plasma Ca followed by subsequent recovery (in lake whitefish but not brown trout) with few mortalities (100% survival for lake whitefish and 93% for brown trout). Both control and exposed fish swam to 85% of the single swim Ucrit and no differences in performance were seen. The Ucrit of unexposed controls in the second swim challenges were not different from the single swim Ucrit. However, second swim performance was significantly reduced in Cd exposed fish, particularly after a week of exposure where 31% and 38% reductions were observed for brown trout and lake whitefish respectively. Swimming to 85% Ucrit resulted in metabolic expenditure with little recovery after 30min. Few differences were observed between control and Cd exposed fish with the exception of a reduction in resting white muscle ATP stores of Cd exposed fish after 1 week of exposure. The results show that chronic sublethal Cd exposure results in an impairment of swimming ability in repeat swim challenges but this impairment is generally not related to metabolic processes in white muscle.

The initial tolerance to sub-lethal Cd exposure is the same among ten naïve pond populations of Daphnia magna, but their micro-evolutionary potential to develop resistance is very different

Genetic variation complicates predictions of both the initial tolerance and the long-term (micro-evolutionary) response of natural Daphnia populations to chemical stressors from results of standard single-clone laboratory ecotoxicity tests. In order to investigate possible solutions to this problem, we aimed to compare the initial sub-lethal tolerance to Cd of 10 naïve natural pond populations of Daphnia magna as well as their evolutionary potential to develop increased resistance. We did so by measuring reproductive performance of 120 clones, i.e. 12 clones hatched from the recent dormant egg bank of each of 10 populations, both in absence (Cd-free control) and presence of 4.4μgCd/L. We show that the initial tolerance, defined as the reproductive performance of individuals of the first generation exposed to Cd relative to that in a Cd-free control was not significantly different among the 10 studied pond populations and averaged 0.82±0.04 over these populations. Moreover, these populations’ initial tolerances were also not significantly different from the mean initial tolerance of 0.87±0.08 at 4.0μgCd/L measured for a group of 7 often-used laboratory clones, collected from a range of European ecotoxicity testing laboratories. This indicates that the initial response of naïve natural pond populations to sub-lethal Cd can be relatively accurately predicted from ecotoxicity test data from only a handful of laboratory clones. We then used estimates of broad-sense heritability of Cd tolerance (H2) – based on the same dataset – as a proxy of these populations’ capacities to evolutionarily respond to Cd in terms of the development of increased resistance, which is here defined as the increase with time of the frequency of clones with a higher Cd tolerance in the population (accompanied with an increase of mean Cd-tolerance of the population above the initial tolerance). We show that the populations’ estimated H2 values of Cd-tolerance cover almost the entire theoretically possible range, ranging from not significantly different from zero (for five populations) to between 0.48 and 0.81 (for the five other populations). This indicates that, unlike the initial tolerance to Cd, the (long-term) micro-evolutionary response to Cd may be very different among natural pond populations. Therefore, we conclude that it may be very difficult to predict the long-term response of an unstudied population to chemical stress from tolerance data on a sample of other populations. It is therefore suggested that new methods for forecasting long-term responses should be explored, such as the development of predictive models based on the combination of population-genomic and tolerance time-series data.

Sublethal Cd-induced cellular damage and metabolic changes in the freshwater crab Sinopotamon henanense

To explore whether sublethal cadmium (Cd) exposure causes branchial cellular damages and affects the metabolic activity in brachyuran crustaceans, the freshwater crab Sinopotamon henanense was exposed to 0.71, 1.43, and 2.86 mg/L Cd(2+) for 3 weeks. Gill morphology, metabolic activity (activities of isocitrate dehydrogenase (IDH), cytochrome c oxidase (CCO) and lactate dehydrogenase (LDH), mRNA expression of CCO active subunit 1 (cco-1) and ldh, as well as ATP levels) in crab muscle were investigated. The results showed that sublethal Cd exposure caused profound morphological damages in the gills. The branchial epithelial cells were disorganized and vacuolized. Ultrastructurally, a decrease in number of apical microvilli, vacuolized mitochondria, and condensed chromatin were observed in gill epithelial cells. Correspondingly, the Cd exposure also induced downregulations of cco-1 and ldh mRNA expression and reduced activities of IDH, CCO, and LDH, in accordance with the lower ATP level in crab muscle. These results led to the conclusion that gill damage caused by sublethal Cd exposure could lead to an impairment of oxygen uptake of S. henanense, and the inhibition of metabolic activity decreases the oxygen demand of the crab and assists them to survive under the condition of lower oxygen availability. These effects add to our understanding on toxic effects of Cd and survival management of S. henanense subchronically exposed to sublethal Cd.

Molecular characterization and expression of a novel homolog of uncoupling protein 5 (UCP5) from the eastern oyster Crassostrea virginica (Bivalvia: Ostreidae)

Uncoupling proteins (UCPs) belong to the mitochondrial anion carrier gene family which has been implicated in diverse physiological functions ranging from thermoregulation to antioxidant defense. In mammals, the UCP family is well characterized and contains five members (UCP1-5). In contrast, invertebrate homologues of uncoupling proteins are much less studied both from the viewpoints of structure and function. In this study we report nucleotide and predicted protein structure of an important member of UCP family, UCP5 from eastern oysters Crassostrea virginica. UCP5 from oysters appears to be a close homolog of the mammalian brain mitochondrial carrier protein (BMCP1, or UCP5) and is the first full-length UCP described from a Lophotrochozoan invertebrate. Evolutionary analysis of UCP sequences indicates at least three monophyletic UCP branches (UCP1-3, UCP4 and UCP5) that have diverged early in the evolution, prior to the divergence of vertebrates and invertebrates. In oysters, two forms of UCP5 transcript are found (UCP5S and UCP5L) that differ by 152 bp in length due to the presence of an intron in UCP5L. UCP5 was expressed in all studied oyster tissues, unlike mammals, where UCP5 is predominantly expressed in brains and male gonads. Hypoxia-reoxygenation stress, sublethal Cd exposure (50 μg L − 1 Cd for 56 days) and acclimation to different temperatures (12 and 20 °C) had no significant effect on UCP5 mRNA expression in oysters indicative of its relative unimportance in antioxidant defense and temperature adaptation of oyster mitochondria. These data suggest that despite the relatively high degree of evolutionary conservation of the UCP5 amino acid sequence, its functional significance in mitochondria changed in the course of evolution of mollusks and vertebrates.

Cadmium accumulation and biochemical responses in Sparus aurata following sub-lethal Cd exposure

Cadmium (Cd), a heavy metal with limited biological function, is widely distributed in the aquatic environment as a result of natural and anthropogenic activities. The effect of 4 and 11 days exposure of gilthead sea bream Sparus aurata to sub-lethal concentrations of Cd was evaluated as levels of Cd content and Cd–metallothionein (MT) presence in different organs. The possible genotoxic effect was also evaluated in erythrocytes by using the “comet assay”, a promising tool for estimating DNA damage at the single-cell level. The results obtained show that in the controls, Cd content was significantly higher in gills compared to in liver, but the treatment of fish with 0.1mg/l Cd induced a stronger accumulation of metal in liver depending on the length of the exposure period. Cd traces were found in plasma, muscle and kidney. Cd forms complexes in the cytosol with MT only in the liver but Cd–MT content significantly increased after 11 days of exposure to the metal, while after 4 days of treatment the protein level was similar to the control. The “comet assay” performed on S. aurata eryhtrocytes isolated from fish treated for 4 and 11 days with 0.1mg/l Cd, showed that there was no DNA damage at both exposure periods.

Cell Death, Stress-Responsive Transgene Activation, and Deficits in the Olfactory System of Larval Zebrafish Following Cadmium Exposure

Cadmium (Cd) is a well-described environmental pollutant known to have adverse effects in fish, including behavioral deficits. We have previously reported the development of an in vivo system that utilizes hsp70 gene activation as a measure of acute 3 h cadmium toxicity in whole living transgenic zebrafish larvae carrying a stably integrated hsp70-enhanced green fluorescent protein (eGFP) reporter gene. Here, we report that activation of this transgene in olfactory epithelium of zebrafish larvae during 96 h sublethal Cd exposure is predictive of cadmium-induced cell death, altered histological and surface organization of the epithelium, and changes in olfactory dependent behavior. The transgene is first activated in the olfactory epithelium at concentrations below those giving rise to significant defects, but exhibits a more robust response following exposure to Cd at concentrations that begin to cause significant cell death, morphological alterations, and behavioral deficits. Further, the data show that Cd-induced olfactory deficits reported previously in juvenile and adult fish can also occur during larval stages of fish development, and that such behavioral deficits are closely associated with cell death and structural alterations in the olfactory epithelium.

Effect of low salinity on cadmium accumulation and calcium homeostasis in the shore crab (Carcinus maenas) at fixed free Cd2+ concentrations.

Increased Cd toxicity at low salinity has been attributed to increased free Cd2+ ion concentration ([Cd2+]sw), but transfer to dilute seawater also stimulates physiological ionic regulation in crabs. In this study, Cd accumulation and Ca homeostasis in the shore crab (Carcinus maenas) were explored at fixed [Cd2+]sw to reveal the physiological events during sublethal Cd exposure. Crabs were exposed to 3.4 or 34 microg/L [Cd2+], in both 100% seawater (SW) and 33% SW for up to 10 d and sampled for hemolymph composition as well as gill and hepatopancreas Ca, Cd, and Ca-ATPase activity. Cadmium exposure ameliorated the expected fall in hemolymph osmotic pressure and NaCl at low salinity and generally protected tissue Ca from decline. Cadmium exposure alone (within salinity) inhibited Ca-ATPase, but this was offset by stimulation of Ca-ATPase at low salinity. The Ca-ATPase activity in the anterior and posterior gills showed different responses to Cd/low salinity stress. Crabs were more sensitive to a 10-fold increase in [Cd2+]sw at low salinity. Overall, we conclude that exposure to a fixed sublethal [Cd2+]sw reveals a compensatory physiological response that is driven primarily by salinity rather than Cd2+ free ion concentration. Physiological responses are therefore important during low-level Cd exposure in dilute seawater.

카드뮴 장기노출 후 넙치, Paralichthys olivaceus의 기관에 따른 카드뮴의 배출

넙치 (P. olivaceus)를 카드뮴 아치사 농도인 5, 50, 100 <TEX>${\mu}g/L$</TEX>의 구간에 30일간 노출시킨후 청장기간을 가져 아가미, 간, 신장, 창자 및 근육에서 축적된 카드뮴의 제거정도를 조사하였다. 아가미에서는 노출농도 50 <TEX>${\mu}g/L$</TEX> 이상에서는 배출 10일째부터 유의한 감소를 나타내었으며, 배출이 가장 빨랐던 기관은 창자로써 배출 10일째부터 노출농도 50, 100 <TEX>${\mu}g/L$</TEX>에서 <TEX>$50\%$</TEX> 이상의 제거율을 나타냈다. 간은 배출 10일째부터 유의한 감소를 나타냈으며, 노출 20일 이후에는 노출구간 50, 100 <TEX>${\mu}g/L$</TEX>에서는 각각 <TEX>$66.20\%$</TEX>와 <TEX>$86.22\%$</TEX>의 제거율을 나타냈다. 신장에는 카드뮴 노출이후 각 구간에서 유의한 감소를 나타내지 않았으며, 근육은 다른 기관들과는 달리 카드뮴 노출 이후 배출실험에서도 지속적인 농도 증가가 나타냈다. 카드뮴 노출 30일 후 배출되는 20일 동안 가장 많이 배출되는 기관의 순서는 창자>간<TEX>${\geq}$</TEX>아가미>>신장의 순으로 나타났다. 아가미, 창자와 간은 배출 20일 동안 급격한 카드뮴의 제거가 나타났으며, 신장은 유의적인 배출이 이루어지지 않고 농도변화가 거의 없었다. 근육은 배출이 이루어지지 않고, 카드뮴이 없는 해수에서도 지속적으로 증가하는 경향을 나타내었다. Experiments were carried out to investigate the elimination of cadmium in tissues (gill, intestine, liver, kidney and muscle) of flounder, Paralichthys olivaceus, after sub-lethal Cd exposure (5, 50, 100 <TEX>${\mu}g/L$</TEX>). During the depuration phase, Cd concentration in the gill decreased immediately following the end of the exposure period. The elimination rates at the end of depuration periods were <TEX>$18\%$</TEX> for 50 <TEX>${\mu}g/L$</TEX> exposure and <TEX>$70\%$</TEX> for 100 <TEX>${\mu}g/L$</TEX> exposure. Intestine showed fastest elimination rates of Cd at all concentration. At the end of the depuration period, the Cd concentration was similar to that in the control, Cd elimination in liver significantly decreased after 10 days of depuration period. After 20 days of depuration, the elimination rate was <TEX>$66.20\%$</TEX> in the fish exposed to 50 <TEX>${\mu}g/L$</TEX> and <TEX>$86.22\%$</TEX> in the fish exposed to 100<TEX>${\mu}g/L$</TEX>. The order of cadmium elimination in tissues were decreased intestine>liver<TEX>${\geq}$</TEX>gill>>kidney during depuration periods. In this study the gill, intestine and liver showed faster elimination rate of Cd in 50, 100 <TEX>${\mu}g/L$</TEX>-Cd exposure concentration. After the end of the Cd exposure, the Cd concentration in the kidney slowly decreased or remained constant and the Cd concentration in the muscle slowly increased or remained constant.

Effects of long term sublethal Cd exposure in rainbow trout during soft water exposure: implications for biotic ligand modelling

The objectives of the study were to determine the physiological and toxicological effects of chronic cadmium exposure on juvenile rainbow trout in soft water. Particular attention focused on acclimation, on comparison to an earlier hard water study, and on whether a gill surface binding model, originally developed in dilute soft water, could be applied in this water quality to fish chronically exposed to Cd. Juvenile rainbow trout, on 3% of body weight daily ration, were exposed to 0 (control), 0.07, and 0.11 μg l −1 Cd [as Cd(NO 3) 2·4H 2O] in synthetic soft water (hardness=20 mg l −1 as CaCO 3, alkalinity=15 mg l −1 as CaCO 3, pH 7.2) for 30 days. Mortality was minimal for all treatments (up to 14% for 0.11 μg l −1 Cd). No significant effects of chronic Cd exposure were seen in growth rate, swimming performance (stamina), routine O 2 consumption, or whole body/plasma ion levels. In contrast to the hard water study, no acclimation occurred in either exposure group in soft water, with no significant increases in 96-h LC 50 values. Cadmium accumulated in a time-dependent fashion to twice the control levels in the gills and only marginally in the liver by 30 days. No significant Cd accumulation occurred in the gall bladder or whole body. Cadmium uptake/turnover tests were run using radioactive 109Cd for acute (3 h) exposures. Saturation of the gills occurred for control fish but not for Cd-exposed fish when exposed to up to 36 μg l −1 Cd for 3 h. Cd-exposed trout accumulated less ‘new’ Cd in their gills compared to controls and they internalized less 109Cd than control fish. This effect of lowered Cd uptake by the gills of acclimated trout was earlier seen for the fish acclimated to 10 μg l −1 Cd in hard water. The affinity of the gill for Cd was greater in hard water (log K Cd-gill=7.6) than in soft water (log K Cd-gill=7.3) but the number of binding sites ( B max=0.20 μg g −1 gill) was similar in both media. In addition, there was a shift in affinity of the gill for Cd (i.e. lowered log K Cd-gill) and increased B max with chronic Cd exposure in both soft water and hard water. We conclude that the present gill modelling approach (i.e. acute gill surface binding model or Biotic Ligand Model) does work for soft and hard water exposures but there are complications when applying the model to fish chronically exposed to cadmium.

Cadmium accumulation, gill Cd binding, acclimation, and physiological effects during long term sublethal Cd exposure in rainbow trout

Juvenile rainbow trout, on 3% of body weight daily ration, were exposed to 0 (control), 3, and 10 μg l −1 Cd (as Cd(NO 3) 2 · 4H 2O) in moderately hard (140 mg l −1 as CaCO 3), alkaline (95 mg l −1 as CaCO 3, pH 8.0) water for 30 days. Particular attention focused on acclimation, and on whether a gill surface binding model, originally developed in dilute softwater, could be applied in this water quality to fish chronically exposed to Cd. Only the higher Cd concentration caused mortality (30%, in the first few days). The costs of acclimation, if any, in our study were subtle since no significant effects of chronic Cd exposure were seen in growth rate, swimming performance (stamina and U Crit), routine O 2 consumption, or whole body ion levels. Substantial acclimation occurred in both exposure groups, manifested as 11- to 13-fold increases in 96-h LC 50 values. In water quality regulations, which are based on toxicity tests with non-acclimated fish only, this remarkable protective effect of acclimation is not taken into account. Cd accumulated in a time- and concentration-dependent fashion to 60–120× (gills), 8–20× (liver), 2–7× (carcass), and 5–12× (whole bodies) control levels by 30 days. Chronically accumulated gill Cd could not be removed by ethylenediaminetetraacetic acid (EDTA) challenge. These gill Cd concentrations were 20- to 40-fold greater than levels predicted by the gill-binding model to cause mortality during acute exposure. In short-term gill Cd-binding experiments (up to 70 μg l −1 exposures for 3 h), gill Cd burden increased as predicted in control fish, but was not detectable against the high background concentrations in acclimated fish. In light of these results, Cd uptake/turnover tests were performed using radioactive 109Cd to improve sensitivity. With this approach, a small saturable binding component was seen, but could not be related to toxic response in acclimated fish. Acclimated trout internalized less 109Cd than control fish, but interpretation was complicated by the possibility of radioisotopic exchange and specific activity dilution in the large `cold' Cd pool on the gills. We conclude that gill Cd burden is not predictive of mortality in acclimated fish, that the present gill modelling approach does not work in acclimated fish, and that longer term 109Cd turnover studies are needed for this purpose.