Subcellular Distribution Of Metals Research Articles

Silicon-Rich Biochar Detoxify Multiple Heavy Metals in Wheat by Regulating Oxidative Stress and Subcellular Distribution of Heavy Metal

Silicon is a quasi-essential trace nutrient for plant growth and is frequently employed to remediate soils of heavy metal pollution in agriculture. However, silicon’s role and mechanism in reducing heavy metal toxicity have not been well understood, especially for multi-heavy metals such as cadmium, zinc, lead, and arsenic (usually treated as a heavy metal). In this study, the effects of different silicon-rich materials (silicate, rice husk biochar (RHB), and RHB + bentonite) on growth trait, antioxidant response, heavy metal accumulation, and distribution of wheat grown in two soils polluted by multiple heavy metals (Cd, Zn, Pb, and As) were investigated. The results revealed that the addition of silicon-rich materials enhanced plant growth, improved the photosynthetic attributes in leaf tissues, and decreased the contents of Cd, Zn, Pb, and As in wheat shoots and grains. The examination of the subcellular distribution of heavy metals in plants implied that silicon-rich materials transferred heavy metals as intracellular soluble fractions to the cell walls, indicating the reduction of mobility and toxicity of heavy metals in the plants. In addition, the application of the silicon-rich materials reduced oxidative damage in plants by downregulating plant antioxidant response systems and decreasing the production of malondialdehyde (MDA), ascorbic acid (AsA), and glutathione (GSH). Moreover, fractionation analysis of soil heavy metals showed that silicon-rich amendments could convert bioavailable heavy metals into immobilized forms. With the comparation of different silicon-rich materials, combined RHB and bentonite could better remediate multi-heavy metal-polluted soils and promote wheat production. The effect of the silicate component was stressed in this paper but some of the potential benefits might have arisen from other components of the biochar.

Do we know the cellular location of heavy metals in seaweed? An up-to-date review of the techniques

Seaweeds are dominant organisms in coastal environments. However, in the context of global change, the integrity of these organisms is threatened by metal pollution. It is therefore important to understand how seaweeds are affected by metal concentrations in the water. Measuring the concentrations of metals in seaweed provides information about the effects of metal pollution on the seaweeds themselves and their ecosystems. Nonetheless, correct interpretation of this type of analysis requires knowledge of the cellular location of the pollutants, as the effects will differ depending on whether the metals are present in particles adhered to the surface, attached to external polysaccharides or dissolved in the cytoplasm. Thus, the objectives of this study were to compile the available information on the subcellular distribution of metals in seaweeds and to conduct a critical review of the information. We found that the existing studies provide contrasting, sometimes contradictory, results. Thus, metals have been detected entirely intracellularly and also mainly outside of the cells. In all of the studies reviewed, which used different techniques (mainly extracellular elution, X-ray microanalysis and centrifugation), methodological and/or conceptual problems were identified that raise questions about the effectiveness of each approach. To obtain reliable information about the distribution of metals in algal cells, further studies must be conducted that take into consideration the differences between elements and algal species and the limits of the methods used to measure the elements.

Significance of chemical affinity on metal subcellular distribution in yellow perch (Perca flavescens) livers from Lake Saint-Pierre (QUEBEC, Canada)

The subcellular partitioning approach provides useful information on the location of metals within cells and is often used on organisms with high levels of bioaccumulation to establish relationships between the internal concentration and the potential toxicity of metals. Relatively little is known about the subcellular partitioning of metals in wild fish with low bioaccumulation levels in comparison with those from higher contaminated areas. This study aims to examine the subcellular partitioning of various metals considering their chemical affinity and essentiality at relatively low contamination levels. Class A (Y, Sr), class B (Cu, Cd, MeHg), and borderline (Fe, Mn) metal concentrations were measured in livers and subcellular fractions of yellow perch (n = 21) collected in Lake Saint-Pierre, QC, Canada. The results showed that all metals, apart from MeHg, were distributed among subcellular fractions according to their chemical affinity. More than 60% of Y, Sr, Fe, and Mn were found in the metal-sensitive fractions. Cd and Cu were largely associated with the metallothionein-like proteins and peptides (60% and 67% respectively) whereas MeHg was found mainly in the metal-sensitive fractions (86%). In addition, the difference between the subcellular distribution of Cu and other essential metals like Fe and Mn denotes that, although the essentiality of some metals is a determinant of their subcellular distribution, the chemical affinity of metals is also a key driver. The similarity of the subcellular partitioning results with previous studies on yellow perch and other fish species from higher contaminated areas supports the idea that metals are distributed in the cellular environment according to their chemical properties regardless of the bioaccumulation gradient.

Synchrotron fluorescence imaging of individual mouse beta-cells reveals changes in zinc, calcium, and iron in a model of low-grade inflammation.

Pancreatic beta-cells synthesize and secrete insulin maintaining an organism's energy homeostasis. In humans, beta-cell dysfunction and death contribute to the pathogenesis of type 2 diabetes (T2D). Although the causes of beta-cell dysfunction are complex, obesity-induced low-grade systemic inflammation plays a role. For example, obese individuals exhibiting increased levels of proinflammatory cytokines IL-6 and IL-1beta have a higher risk of beta-cell dysfunction and T2D. Interestingly, obesity-induced inflammation changes the expression of several cellular metal regulating genes, prompting this study to examine changes in the beta-cell metallome after exposure to proinflammatory-cytokines. Primary mouse beta-cells were exposed to a combination of IL-6 and IL-1beta for 48 hours, were chemically fixed and imaged by synchrotron X-ray fluorescent microscopy. Quantitative analysis showed a surprising 2.4-fold decrease in the mean total cellular content of zinc from 158± 57.7 femtograms (fg) to 65.7± 29.7 fg; calcium decreased from 216± 67.4 to 154.3± 68.7 fg (control vs. cytokines, respectively). The mean total cellular iron content slightly increased from 30.4± 12.2 to 47.2± 36.4 fg after cytokine treatment; a sub-population of cells (38%) exhibited larger increases of iron density. Changes in the subcellular distributions of zinc and calcium were observed after cytokine exposure. Beta-cells contained numerous iron puncta that accumulated still more iron after exposure to cytokines. These findings provide evidence that exposure to low levels of cytokines is sufficient to cause changes in the total cellular content and/or subcellular distribution of several metals known to be critical for normal beta-cell function.

Does combined heavy metal stress enhance iron plaque formation and heavy metal bioaccumulation in Kandelia obovata?

Iron plaque (IP) has significant impact on rhizosphere environment, and affects the uptake and translocation of heavy metals in plants. However, information on IP formation and heavy metal bioaccumulation for mangrove plants under combined heavy metal stress is currently limited. Based on a 120-day pot experiment, this study explored the temporal changes of IP formation on root surface of Kandelia obovata, and heavy metal bioaccumulations under combined heavy metal stress of Cu, Pb and Zn. The results showed that IP formations on root tip, middle and basal roots were improved with combined heavy metal stress and treatment time (root tip: 0.49−0.61 mg g−1 to 2.55–4.77 mg g−1; middle root: 0.27−0.31 mg g−1 to 3.01–4.22 mg g−1; basal root: 0.16−0.23 mg g−1 to 2.24–4.10 mg g−1). IP formation was accompanied with improved depositions of Cu and Pb on root surface, regardless of root tip, middle and basal roots. IP and roots deposited large amounts of Cu (24.97–69.52 μg g-1, 9.39–154.75 μg g−1), Pb (8.35–84.53 μg g-1, 12.29–191.49 μg g-1) and Zn (85.71–139.19 μg g-1, 51.07–195.65 μg g−1), inhibiting their translocations to aboveground leaves. Based on subcellular distribution and chemical forms of heavy metals in roots, the improved depositions of phosphate-integrated Cu (6.77 %–25.13 %), oxalate-integrated Pb (34.89 %–57.88 %), as well as pectate- and protein-integrated Zn (14.22 %–35.41 %) were detected in root cell vacuoles. In conclusion, the combined heavy metal stress enhanced IP formation and heavy metals deposition (especially for Cu and Pb) on root surface, and improved their vacuolar sequestration and chelation in roots of K. obovata.

Roles of subcellular metal homeostasis in crop improvement

Improvement of crop production in response to rapidly changing environmental conditions is a serious challenge facing plant breeders and biotechnologists. Iron (Fe), zinc (Zn), manganese (Mn), and copper (Cu) are essential micronutrients for plant growth and reproduction. These minerals are critical to several cellular processes including metabolism, photosynthesis, and cellular respiration. Regulating the uptake and distribution of these minerals could significantly improve plant growth and development, ultimately leading to increased crop production. Plant growth is limited by mineral deficiency, but on the other hand, excess Fe, Mn, Cu, and Zn can be toxic to plants; therefore, their uptake and distribution must be strictly regulated. Moreover, the distribution of these metals among subcellular organelles is extremely important for maintaining optimal cellular metabolism. Understanding the mechanisms controlling subcellular metal distribution and availability would enable development of crop plants that are better adapted to challenging and rapidly changing environmental conditions. Here, we describe advances in understanding of subcellular metal homeostasis, with a particular emphasis on cellular Fe homeostasis in Arabidopsis and rice, and discuss strategies for regulating cellular metabolism to improve plant production.

Differences in the subcellular distribution of cadmium and copper in the gills and liver of white sturgeon (Acipenser transmontanus) and rainbow trout (Oncorhynchus mykiss)

Recent studies have shown that white sturgeon (Acipenser transmontanus) are more resistant to cadmium (Cd) compared to rainbow trout (Oncorhynchus mykiss), whereas they are more sensitive than rainbow trout when exposed to copper (Cu). Differences in the subcellular distribution of metals among species could be one of the factors responsible for the differences in the sensitivity to metals. Although, subcellular distribution has been studied extensively in many species with many metals, its direct role in species-specific differences in the sensitivity has not been well studied. The objective of this study was to evaluate the role of subcellular distribution of metals in species-specific differences in the sensitivity to metals between sturgeon and trout. We compared the subcellular distribution of metals Cd and Cu in the cellular debris, heat-stable proteins, heat-denatured fraction, metal-rich granules, and organelles fractions from the gills and liver after exposure of juveniles of both species to 1.25 and 20 μg/L Cd and Cu for 8 days, respectively. Sturgeon diverted a higher amount of Cd towards biologically inactive metal pool (BIM) and a lower amount towards the biologically active metal pool (BAM) compared to trout in both tissues. This explained why sturgeon are able to tolerate a relatively higher exposure level to Cd compared to trout. For Cu, there was no statistically significant species-specific differences in the amounts diverted towards either BAM or BIM; hence, white sturgeon’s greater sensitivity to Cu was not explained by its subcellular distribution strategies.

Subcellular metal distribution in two deep-sea mollusks: Insight of metal adaptation and detoxification near hydrothermal vents

In this study, we determined the concentrations of Cu, Zn, Ni, Cd, Pb and As and their subcellular distributions within the tissues of mussels (Bathymodiolus marisindicus) and snails (Gigantopelta aegis) from two hydrothermal vent regions, i.e., Tiancheng and Longqi, at Southwest Indian Ridge. Mussels collected from the two venting regions showed comparable concentrations for Ni and Pb, but Cu, Zn, Cd and As concentrations were significantly different in mussel gills between the two vent regions. Similar ranges of metal concentrations were found in the snails as those in the mussels, but most of the metals were mainly accumulated in the viscera, except for Ni. Similar subcellular partitioning of Cu, Zn and Cd was documented in different mussel tissues, with cellular debris (50%) being the predominant fraction, followed by equivalent values in other fractions. Lead was distributed in both cellular debris and metal-rich granules (MRG) fraction, whereas Ni was predominantly distributed in MRG (90%). Arsenic was mainly partitioned in cellular debris and metallothionein-like protein. However, deep-sea snails displayed elevated subcellular partitioning of Cu in the organelles (up to 60%) and may be more susceptible to Cu stress than the mussels. Our results demonstrated the metal-specificity of detoxification strategies in these deep-sea hydrothermal vent mollusks, and the mussels may be more adaptable to high metal exposures than the snails at hydrothermal vent.

Integrated assessment of the acclimation capacity of the marine bivalve Crenomytilus grayanus under naturally highly contaminated conditions: Subcellular distribution of trace metals and structural alterations of nephrocytes

The aim of our work was to assess whether the cellular processes in the nephrocytes of the long-lived mussel Crenomytilus grayanus tend to acclimation or destruction under trace metal contamination. Mussels were collected from three sites in the north-western Pacific Ocean: reference site, upwelling site, and a site highly contaminated with trace metals. Concentration, subcellular distribution of trace metals (Cd, Cu, Zn, and Pb) in the mussel kidneys, and ultrastructural alterations of the nephrocytes were studied. To assess the total load of accumulated trace metals, the total concentration coefficient (∑СС) was determined.In the kidneys of the reference C. grayanus, trace metals were eliminated from cell metabolism mainly by lysosomal granules or residue bodies. Under high levels of contamination, the defense mechanisms of C. grayanus are practically suppressed (no metallothionein-like protein peak, decreased content of granules) by the total effect of accumulated pollutants that leads to the destruction of cellular structures. Under natural conditions (upwelling site), increased accumulation of trace metals in the mussel kidneys did not lead to an increase in the number or size of lysosomal granules. However, abnormal high Cd accumulation in the kidneys caused the synthesis of high levels of metallothionein-like proteins that sequester most of the studied trace metals. To quickly lower the metal levels in nephrocytes under these conditions, a unique long-term acclimatory response — apocrine-like secretion in nephrocytes, which provides rapid elimination of me—MTLP complexes from the cell arose. Thus, our integrated study of the subcellular distribution of trace metals and ultrastructural alterations in nephrocytes allowed us to characterize the features of the structural and functional alterations in mussel cells under the field conditions tested.

Thermal discharge influences the bioaccumulation and bioavailability of metals in oysters: Implications of ocean warming

Human-induced temperature changes influence coastal regions, both via thermal pollution and ocean warming, which exerts profound effects on the chemistry of metals and the physiology of organisms. However, it remains unknown whether the increased temperature of discharged water or ocean warming, as a result of climate change, lead to an increase of human health risks associated with the consumption of sea foods. In this study, the influence of temperature on metal accumulation by oysters was studied in individuals collected from a coastal area affected by the thermal water discharge of the Houshi Power Plant, China. The bioaccumulation factor (BAF) and oral bioavailability (OBA) of metals in oysters was determined. Elevated temperatures led to an increase in BAF for Cu, Zn, Hg, and Cd (p < 0.05), but no change was observed for As and Pb (p > 0.05). The OBA for Cd, As, and Pb correlated positively to elevated temperatures (p < 0.05). However, for Cu and Zn, OBA was negatively correlated with increasing temperature (p < 0.05). As, Pb, and Cd in the trophically available metal (defined as a sum of heat-stable proteins, heat-denaturable proteins, and organelles) was significantly elevated at the highest temperature seawater site (site A) compared to the lowest seawater site (site B). Thus, the irregular variation of OBA for each metal may be the result of variations in the subcellular distribution of metals and the protein quality influenced by the increased temperature. Moreover, the increased temperature and increased the hazard quotient values of As and Cd (p < 0.05 for As, n = 6, p < 0.05 for Cd, n = 6), which provided an indication of the potential risks of the consumption of oysters or other seafood to future warming under climate change scenarios.

Biokinetics and subcellular distribution of metals in Daphnia magna following Zn exposure: Implication for metal regulation

Although many studies have addressed the effects of various physicochemical and biological factors on metal bioaccumulation in aquatic organisms, the influences of metal exposure history have drawn much less attention. In the present study, we investigated the effects of different Zn exposure regimes (concentration: 1–200 μg/L, duration: 1–7 d) on the subsequent biokinetics, metallothionien-like protein (MTLP) induction and subcellular distribution of Cd and Zn in Daphnia magna. Zn body burden increased significantly with elevated exposure concentrations, but was kept within a narrow range regardless of the 200-fold variation of Zn concentrations. Significant induction (7–14 folds) of MTLP by Zn exposure was evident, but was decoupled from the Zn body accumulation. Under different regimes of Zn exposure, Zn was evenly distributed in insoluble (cellular debris, intracellular organelles and metal-rich granules) and soluble fractions (heat sensitive protein and MTLP). However, >60% of Cd was bound with MTLP regardless of the exposure concentration and duration. The biokinetic processes including uptake from the dissolved phase, food assimilation and excretion of Cd and Zn were quantified with radioactive tracers. The uptake rate constants of Cd and Zn from the dissolved phase either remained comparable or increased following Zn pre-exposure, and the efflux of Zn increased by nearly two-fold to counteract the increased uptake from water. The dietary assimilation of Zn also decreased significantly in response to Zn exposure. However, Cd assimilation efficiency was kept relatively constant in each Zn pre-exposure regime. Our results showed that Zn exposure had significant influences on the biokinetics and physiology of daphnids. Daphnids attempted to maintain the Zn body burden within a narrow range by modification of biokinetic processes instead of subcellular distribution.

Accumulation and subcellular distribution of heavy metal in Paulownia fortunei cultivated in lead-zinc slag amended with peat

The contamination of toxic heavy metals was a major issue of concern in the last century. A fast-growing metal-accumulating woody plant is a promising approach for the remediation of toxic heavy metal. In this study, the transportation of heavy metals (Pb, Zn, Cu, and Cd) in Paulownia fortunei cultivated in lead-zinc slag amended with different mass ratios of peat (CK: 0; T1: 10%; T2: 20%; T3: 30%) was investigated, as well as the subcellular distribution of Pb, Zn, Cu, and Cd in Paulownia fortunei. The results showed that the accumulation content of Pb, Zn, Cu, and Cd in Paulownia fortunei were increased with peat amendment, which was in the range of 4.216 ∼ 6.853, 20.905 ∼ 23.017, 1.898 ∼ 2.572, and 0.530 ∼ 0.616 mg/pot, respectivly. The experimental group with 30% dose of peat showed the best performance on the accumulation content of Pb, Zn, Cu, and Cd, with increase rates (compared to control) of 4.088, 10.573, 1.360, and 0.294 mg/pot, respectively. The bioconcentration, translocation and transfer quantity factor of Pb, Zn, Cu, and Cd were less than 1. Fixation of cell wall and compartmentalization of vacuolar appeared to play an important role in reducing the toxicity of Pb, Zn, Cu, and Cd.

Synergistic effects of chromium and copper on photosynthetic inhibition, subcellular distribution, and related gene expression in Brassica napus cultivars.

Nowadays, modern plant physiology focuses on complex behavior of metal co-contaminants in agrosystems. Keeping this in view, the current study was conducted to investigate the response of two Brassica napus cultivars (Zheda 622 and ZS 758) under co-contamination of copper (Cu2+) and chromium (Cr6+) to observe their effects on plant growth, photosynthetic parameters, and subcellular distribution of these metals in leaves and roots. The results showed that exposure to Cu and Cr causes decline in plant growth, including biomass and plant height. Significant decrease in pigment concentration and the photosynthetic activity [photosynthetic rate (Pn), stomatal conductance (Gs), and transpiration rate (E), maximal quantum yield of photosystem II (Fv/Fm)] in leaves was also observed. Results of subcellular distribution of metals showed that Cu and Cr were predominantly distributed in cell wall and soluble fraction of roots and leaves. Moreover, Cu and Cr in cellular fractions showed a synergistic accumulation pattern under combined metal stress treatment. Both cultivars showed increased levels of reactive oxygen species (ROS), i.e., hydrogen peroxide (H2O2) and superoxide radical (O2•-), and significant modulation in the activities of antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX)] under Cu/Cr alone or their combined treatments. Similarly, expression levels of defense-related genes, such as BnCat, BnApx, BnPrx, and BnSod, were also generally up-regulated compared with control. Electron micrographs (TEM) of the mesophyll and root tip cells indicated prominent alterations both in cellular and organelle levels. Additionally, Cr was found to be more toxic than Cu but less than their combined effect, as revealed by enhanced production of oxidative stress and a reduction in biomass production and photosynthetic activity. The present results also suggest that cultivar ZS 758 is more resistant to Cu/Cr than Zheda 622, due to better adapted metabolism and maintenance of structural integrity under metal stress.

Response of indigenous Cd-tolerant electrochemically active bacteria in MECs toward exotic Cr(VI) based on the sensing of fluorescence probes

Electrochemically active bacteria (EAB) on the cathodes of microbial electrolysis cells (MECs) can remove metals from the catholyte, but the response of these indigenous EAB toward exotic metals has not been examined, particularly from the perspective of the co-presence of Cd(II) and Cr(VI) in a wastewater. Four known indigenous Cd-tolerant EAB of Ochrobactrum sp X1, Pseudomonas sp X3, Pseudomonas delhiensis X5, and Ochrobactrum anthropi X7 removed more Cd(II) and less Cr(VI) in the simultaneous presence of Cd(II) and Cr(VI), compared to the controls with individual Cd(II) or single Cr(VI). Response of these EAB toward exotic Cr(VI) was related to the associated subcellular metal distribution based on the sensing of fluorescence probes. EAB cell membrane harbored more cadmium than chromium and cytoplasm located more chromium than cadmium, among which the imaging of intracelluler Cr(III) ions increased over time, contrary to the decreased trend for Cd(II) ions. Compared to the controls with single Cd(II), exotic Cr(VI) decreased the imaging of Cd(II) ions in the EAB at an initial 2 h and negligibly affected thereafter. However, Cd(II) diminished the imaging of Cr (III) ions in the EAB over time, compared to the controls with individual Cr(VI). Current accelerated the harboring of cadmium at an initial 2 h and directed the accumulation of chromium in EAB over time. This study provides a viable approach for simultaneously quantitatively imaging Cd(II) and Cr (III) ions in EAB and thus gives valuable insights into the response of indigenous Cd-tolerant EAB toward exotic Cr(VI) in MECs.

Subcellular partitioning of metals and metalloids (As, Cd, Cu, Se and Zn) in liver and gonads of wild white suckers (Catostomus commersonii) collected downstream from a mining operation

In the present study, we examined the subcellular distribution of metals and metalloids (As, Cd, Cu, Se and Zn) in the liver and gonads of wild white suckers (Catostomus commersonii) collected downstream from a metal mining operation (exposure area) and in a reference area. Metal partitioning among potentially metal-sensitive fractions (heat-denatured proteins (HDP), mitochondria and microsomes) and potentially biologically detoxified fractions (heat-stable proteins (HSP) and metal-rich granules) within cells was determined after differential centrifugation, NaOH digestion and heat-denaturation steps. Metal-handling strategies between liver and gonads, and between sexes, were examined. Hepatic metal concentrations were significantly higher in exposed compared to reference fish, especially for Se (14x), Cd (5x) and Cu (3x), and did not vary between sexes. In contrast, gonadal Cd, Cu, Se and Zn concentrations were consistently lower in testes than in ovaries; marked differences in Cd and Se concentrations between exposed and reference fish were observed for both sexes. Overall, metal-handling strategies were similar in both liver (male and female pooled) and female gonads, but differed from those in male gonads, likely due to the different functions assigned to ovaries and testes. Subcellular partitioning of As, Cd and Cu showed that the HSP fraction was most responsive to increased metal exposure, presumably reflecting Cu regulation, and possibly Cd and As detoxification. Zinc concentrations were tightly controlled and mainly found in the HDP fraction. Interestingly, changes in Cd-handling strategy in female gonads were particularly evident, with Cd shifting dramatically from the metal-sensitive HDP fraction in reference fish to the metal-detoxified HSP fraction in exposed fish. It seems that Cd detoxification in female gonads was not fully induced in the less contaminated fish, but became more effective above a threshold Cd concentration of 0.05 nmol/g dry weight. Partitioning of Se was different, with the largest contributor to the total liver and gonad Se burdens being the putative metal-sensitive HDP fraction, suggesting that excess Se in this fraction in exposed fish may lead to Se-related stress. The present subcellular partitioning results demonstrate that metal handling strategies vary among metals, between organs and (in some cases) as a function of metal exposure. They also show promise in identifying metals of potential concern in a risk assessment context.

Effects of elevated CO2 levels on subcellular distribution of trace metals (Cd and Cu) in marine bivalves

Hypercapnia (elevated CO2 levels) and pollution with trace metals such as Cu and Cd are common stressors in estuarine habitats that can negatively affect physiology and health of marine organisms. Hypercapnia can modulate toxicity of trace metals including Cu and Cd; however, the physiological and cellular mechanisms of the metal-CO2 interactions are not well understood. We investigated the effects of elevated PCO2 (∼800 and 2000μatm) and metal exposure (50μgl−1 of Cu or Cd) on subcellular distribution of metals in two common species of marine bivalves, Eastern oysters Crassostrea virginica and hard shell clams Mercenaria mercenaria. Oysters accumulated higher burdens of Cu and Cd in the gill tissues compared to clams. In both studied species, Cu was predominantly associated with the metabolically active cell compartments (mitochondria, lysosomes, microsomes and cytosolic enzymes), with a modest fraction sequestered by metallothioneins (∼30%) and the insoluble metal-containing granules (MCG) (∼15–20%). Unlike Cu, Cd was largely sequestered by metallothioneins (∼60–70%), with a relatively small fraction associated with the organelles and the cytosolic enzymes. Mitochondria were the main intracellular target for trace metals accumulating higher concentrations of Cd (and in the case of oysters – of Cu) than other organelles or cytosolic enzymes. Cu accumulation in the metabolically active cellular compartments was independent of the CO2 levels, while Cd content of the organelles and cytosolic enzymes increased at elevated PCO2 in both studied species indicating that hypercapnia may enhance cellular toxicity of Cd in bivalves. Hypercapnia suppressed the sequestration capacity of metallothioneins for Cu and Cd in oysters but increased Cu and Cd load in clam metallothioneins. Thus, metal-induced metabolic injury in oysters may be exaggerated by hypercapnia which enhances metal accumulation in the potentially sensitive intracellular fractions and suppresses the metal detoxification capacity. In contrast, clams appear to be more resistant to the combined effects of hypercapnia and metal exposure reflecting more efficient and robust detoxification mechanisms of this species.

Subcellular Distribution and Chemical Forms of Heavy Metals in Three Types of Compositae Plants from Lead-Zinc Tailings Area

Field investigation on the content of heavy metals in soils and three types of widely distributing compositae plants(Artemisia lavandulaefolia, Ageratum conyzoides L., Crassocephalum crepidioides) in lead-zinc tailings farmland of Yangshuo, Guangxi Zhuang Autonomous Region was carried out, and the differential centrifugation technique and sequential chemical extraction method were used to study the subcellular distribution and chemical forms of heavy metals in these plants. The results indicated that the soil in the tailings farmland was highly contaminated by Cd, Pb and Zn, and their concentrations were 37.7, 5.7 and 8.9 times higher than their respective values of national standard for soil environment quality(GradeⅡ). The contents of Cd, Pb and Zn in the analyzed plants exceeded the normal ranges. Ageratum conyzoides L. and Crassocephalum crepidioides showed strong capability in tolerance, accumulation and transport of Cd, and they could be used as pioneer plants for Cd-phytoremediation in study area and some related areas. In addition, Cd concentrations in the stem and leave of Crassocephalum crepidioides were 159.6 mg·kg-1 and 219.5 mg·kg-1, respectively, which exceeded the threshold of Cd hyperaccumulator. Thus, Crassocephalum crepidioides can be regarded as a Cd-hyperaccumulator. Majority of total Cd, Pb and Zn were found in soluble fraction and cell wall in the plants, while only a small quantity were distributed in organelles. As for chemical forms distribution, Cd, Zn and Pb predominated NaCl-, HAC-, and HCl-extractable forms both in roots and shoots of the plants. Therefore, cell wall binding, vacuolar compartmentalization and distribution mainly in lower active chemical forms were supposed to be the main mechanisms for tolerance to heavy metals in the study plants. A difference of Cd subcellular distribution and its chemical forms in the three Compositae plants was observed. Compared with the shoot of Artemisia lavandulaefolia, more Cd was located in the vacuolus and cellular soluble parts in Ageratum conyzoides L. and Crassocephalum crepidioides. Moreover, the proportion of active chemical Cd in the shoots was lower than that in the roots. These results showed that the subcellular distribution and chemical forms of Cd were related to the plant species,and also indicated Ageratum conyzoides L. and Crassocephalum crepidioides were likely to possess a higher tolerance and accumulation of Cd.

Stimulus-Responsive Prochelators for Manipulating Cellular Metals.

Metal ions are essential for a wide range of physiological processes, but they can also be toxic if not appropriately regulated by a complex network of metal trafficking proteins. Intervention in cellular metal distribution with small-molecule or peptide chelating agents has promising therapeutic potential to harness metals to fight disease. Molecular outcomes associated with forming metal-chelate interactions in situ include altering the concentration and subcellular metal distribution, inhibiting metalloenzymes, enhancing the reactivity of a metal species to elicit a favorable biological response, or passivating the reactivity of a metal species to prevent deleterious reactivity. The systemic administration of metal chelating agents, however, raises safety concerns due to the potential risks of indiscriminate extraction of metals from critical metalloproteins and inhibition of metalloenzymes. One can estimate that chelators capable of complexing metal ions with dissociation constants in the submicromolar range are thermodynamically capable of extracting metal ions from some metalloproteins and disrupting regular function. Such dissociation constants are easily attainable for multidentate chelators interacting with first-row d-block metal cations in relevant +1, + 2, and +3 oxidation states. To overcome this challenge of indiscriminate metal chelation, we have pursued a prodrug strategy for chelating agents in which the resulting "prochelator" has negligible metal binding affinity until a specific stimulus generates a favorable metal binding site. The prochelator strategy enables conditional metal chelation to occur preferentially in locations affected by disease- or therapy-associated stimuli, thereby minimizing off-target metal chelation. Our design of responsive prochelators encompasses three general approaches of activation: the "removal" approach operates by eliminating a masking group that blocks a potential metal chelation site to reveal the complete binding site under the desired conditions; the molecular "switch" approach involves a reversible conformational change between inactive and active forms of a chelator with differential metal binding affinity under specific conditions; and the "addition" approach adds a new ligand donor arm to the prochelator to constitute a complete metal chelation site. Adopting these approaches, we have created four categories of triggerable prochelators that respond to (1) reactive oxygen species, (2) light, (3) specific enzymes, and (4) biological regulatory events. This Account highlights progress from our group on building prochelators that showcase these four categories of responsive metal chelating agents for manipulating cellular metals. The creation and chemical understanding of such stimulus-responsive prochelators enables exciting applications for understanding the cell biology of metals and for developing therapies based on metal-dependent processes in a variety of diseases.

Regulating Subcellular Metal Homeostasis: The Key to Crop Improvement.

Iron (Fe), zinc (Zn), manganese (Mn), and copper (Cu) are essential micronutrient mineral elements for living organisms, as they regulate essential cellular processes, such as chlorophyll synthesis and photosynthesis (Fe, Cu, and Mn), respiration (Fe and Cu), and transcription (Zn). The storage and distribution of these minerals in various cellular organelles is strictly regulated to ensure optimal metabolic rates. Alteration of the balance in uptake, distribution, and/or storage of these minerals severely impairs cellular metabolism and significantly affects plant growth and development. Thus, any change in the metal profile of a cellular compartment significantly affects metabolism. Different subcellular compartments are suggested to be linked through complex retrograde signaling networks to regulate cellular metal homeostasis. Various genes regulating cellular and subcellular metal distribution have been identified and characterized. Understanding the role of these transporters is extremely important to elaborate the signaling between various subcellular compartments. Moreover, modulation of the proteins involved in cellular metal homeostasis may help in the regulation of metabolism, adaptability to a diverse range of environmental conditions, and biofortification. Here, we review progress in the understanding of different subcellular metal transport components in plants and discuss the prospects of regulating cellular metabolism and strategies to develop biofortified crop plants.

Subcellular partitioning profiles and metallothionein levels in indigenous clams Moerella iridescens from a metal-impacted coastal bay

In this study, the effect of environmental metal exposure on the accumulation and subcellular distribution of metals in the digestive gland of clams with special emphasis on metallothioneins (MTs) was investigated. Specimens of indigenous Moerella iridescens were collected from different natural habitats in Maluan Bay (China), characterized by varying levels of metal contamination. The digestive glands were excised, homogenized and six subcellular fractions were separated by differential centrifugation procedures and analyzed for their Cu, Zn, Cd and Pb contents. MTs were quantified independently by spectrophotometric measurements of thiols. Site-specific differences were observed in total metal concentrations in the tissues, correlating well with variable environmental metal concentrations and reflecting the gradient trends in metal contamination. Concentrations of the non-essential Cd and Pb were more responsive to environmental exposure gradients than were tissue concentrations of the essential metals, Cu and Zn. Subcellular partitioning profiles for Cu, Zn and Cd were relatively similar, with the heat-stable protein (HSP) fraction as the dominant metal-binding compartment, whereas for Pb this fraction was much less important. The variations in proportions and concentrations of metals in this fraction along with the metal bioaccumulation gradients suggested that the induced MTs play an important role in metal homeostasis and detoxification for M. iridescens in the metal-contaminated bay. Nevertheless, progressive accumulation of non-essential metals (Cd, and especially Pb) resulting from “spillover” was observed in putative metal- sensitive (e.g., mitochondria and heat-denaturable protein (HDP)) or lysosome/microsome fractions, demonstrating that metal detoxification was incomplete and increased the toxicological risk to M. iridescens inhabiting the metal-impacted environments. Through multiple stepwise regression analysis, the induction of MTs was statistically correlated with the HSP concentrations of Cu, and to a lesser extent with Zn, and ultimately to the Cd concentrations, exhibiting significant dose-dependent relationships. Overall, these findings not only revealed the fates of accumulated metals, but scientifically favored an improved understanding of the detoxification at the subcellular level in response to metal accumulation, supporting the focus of metabolic availability assessment on the intracellular processes or events occurring within organisms.