Intracellular Cadmium Research Articles

Cadmium Transport in Maize Root Segments Using a Classical Physiological Approach: Evidence of Influx Largely Exceeding Efflux in Subapical Regions.

The bidirectional fluxes of cadmium and calcium across the plasma membrane were assessed and compared in subapical maize root segments. This homogeneous material provides a simplified system for investigating ion fluxes in whole organs. The kinetic profile of cadmium influx was characterized by a combination of a saturable rectangular hyperbola (Km = 30.15) and a straight line (k = 0.0013 L h-1 g-1 fresh weight), indicating the presence of multiple transport systems. In contrast, the influx of calcium was described by a simple Michaelis-Menten function (Km = 26.57 µM). The addition of calcium to the medium reduced cadmium influx into the root segments, suggesting a competition between the two ions for the same transport system(s). The efflux of calcium from the root segments was found to be significantly higher than that of cadmium, which was extremely low under the experimental conditions used. This was further confirmed by comparing cadmium and calcium fluxes across the plasma membrane of inside-out vesicles purified from maize root cortical cells. The inability of the root cortical cells to extrude cadmium may have driven the evolution of metal chelators for detoxifying intracellular cadmium ions.

Cadmium Accumulation Involves Synthesis of Glutathione and Phytochelatins, and Activation of CDPK, CaMK, CBLPK, and MAPK Signaling Pathways in Ulva compressa.

In order to analyze the effect of cadmium in Ulva compressa (Chlorophyta), the alga was cultivated with 10, 25, and 50 μM of cadmium for 7 days, and the level of intracellular cadmium was determined. Intracellular cadmium showed an increase on day 1, no change until day 5, and an increase on day 7. Then, the alga was cultivated with 10 μM for 7 days, and the level of hydrogen peroxide, superoxide anions, and lipoperoxides; activities of antioxidant enzymes ascorbate peroxidase (AP), dehydroascorbate reductase (DHAR), and glutathione reductase (GR); the level of glutathione (GSH) and ascorbate (ASC); and the level of phytochelatins (PCs) and transcripts encoding metallothioneins (UcMTs) levels were determined. The level of hydrogen peroxide increased at 2 and 12 h, superoxide anions on day 1, and lipoperoxides on days 3 to 5. The activities of AP and GR were increased, but not the DHAR activity. The level of GSH increased on day 1, decreased on day 3, and increased again on day 5, whereas ASC slightly increased on days 3 and 7, and activities of enzymes involved in GSH and ASC synthesis were increased on days 3 to 7. The level of PC2 and PC4 decreased on day 3 but increased again on day 5. The level of transcripts encoding UcMT1 and UcMT2 increased on days 3 to 5, mainly that of UcMT2. Thus, cadmium accumulation induced an oxidative stress condition that was mitigated by the activation of antioxidant enzymes and synthesis of GSH and ASC. Then, the alga cultivated with inhibitors of calcium-dependent protein kinases (CDPKs), calmodulin-dependent protein kinases (CaMKs), calcineurin B-like protein kinases (CBLPKs), and MAPKs and 10 μM of cadmium for 5 days showed a decrease in intracellular cadmium and in the level of GSH and PCs, with the four inhibitors, and in the level of transcripts encoding UcMTs, with two inhibitors. Thus, CDPKs, CaMK, CBLPKS, and MAPKs are involved in cadmium accumulation and GSH and PC synthesis, and GSH and PCs and/or UcMTs may participate in cadmium accumulation.

Sensitive and Specific Cadmium Biosensor Developed by Reconfiguring Metal Transport and Leveraging Natural Gene Repositories.

Whole-cell biosensors are useful for monitoring heavy metal toxicity in public health and ecosystems, but their development has been hindered by intrinsic trade-offs between sensitivity and specificity. Here, we demonstrated an effective engineering solution by building a sensitive, specific, and high-response biosensor for carcinogenic cadmium ions. We genetically programmed the metal transport system of Escherichia coli to enrich intracellular cadmium ions and deprive interfering metal species. We then selected 16 cadmium-sensing transcription factors from the GenBank database and tested their reactivity to 14 metal ions in the engineered E. coli using the expression of the green fluorescent protein as the readout. The resulting cadmium biosensor was highly specific and showed a detection limit of 3 nM, a linear increase in fluorescent intensities from 0 to 200 nM, and a maximal 777-fold signal change. Using this whole-cell biosensor, a smartphone, and low-tech equipment, we developed a simple assay capable of measuring cadmium ions at the same concentration range in irrigation water and human urine. This method is user-friendly and cost-effective, making it affordable to screen large amounts of samples for cadmium toxicity in agriculture and medicine. Moreover, our work highlights natural gene repositories as a treasure chest for bioengineering.

Effects of Moringa oleifera leaf extracts and its bioactive compound gallic acid on reducing toxicities of heavy metals and metalloid in Saccharomyces cerevisiae

Moringa oleifera leaf extract is rich in antioxidants and has high potential for use to alleviate metal toxicity. Previously, we have reported the roles of aqueous M. oleifera leaf extract in mitigating intracellular cadmium (Cd) accumulation and Cd-induced oxidative stress. In this study, we investigated the protective role of aqueous and/or ethanolic M. oleifera leaf extracts (AMOLE and/or EMOLE) against other metal(loid)s in the eukaryotic model Saccharomyces cerevisiae. Our results show that only the AMOLE remarkably promoted the growth of yeast cells grown in the presence of arsenite (As(III)), Cd, nickel (Ni), and lead (Pb). Although the AMOLE contained lower amount of total phenolic and flavonoid contents and displayed lower DPPH scavenging capacity than the EMOLE, both AMOLE and EMOLE had the same capacity for reducing intracellular ROS levels in yeast cells exposed to As(III), Cd, Ni, and Pb. Moreover, the AMOLE was more effective than the EMOLE in inhibiting intracellular accumulation of these toxic metal(loid)s. In addition, we found that gallic acid, one of important phenolic constituents present in both extracts, could protect yeast cells against As(III) toxicity, likely through its role in decreasing As(III) accumulation and As(III)-induced ROS production. Furthermore, the hydroxyl and carboxyl groups of gallic acid appear to play a critical role in chelating As(III). The present study suggests the promising applications of the AMOLE (and also gallic acid) as protective agents against hazardous metal(loid)s.

Identification of intracellular cadmium transformation in HepG2 and MCF-7 cells

It is of significance to elucidate or understand the intracellular transformation & migration behaviors of heavy metals in specific cells. Herein, we report the fast and efficient separation of cadmium-metallothioneins (Cd-MTs) and Cd2+in cell lysate by a short column capillary electrophoresis (SC-CE), followed by coupling with inductively coupled plasma mass spectrometry (ICP-MS) to facilitate the speciation of intracellular cadmium species. The incorporation of sodium dodecyl sulfate (SDS) in running buffer significantly reduces the peak width of Cd2+from 170 s to 26 s in the electrophoretogram, causing a 5.3-fold improvement on the sensitivity. Linear ranges of 0.5–50 mg L−1,0.056–5.6 mg L−1 and 0.1–10 mg L−1 are achieved for MTs, Cd-MTs (Cd) and Cd2+, respectively, along with detection limits of 0.013 mg L−1 for Cd-MTs (Cd) and 0.020 mg L−1 for Cd2+. The transformation of cadmium in HepG2 and MCF-7 cells is evaluated after their incubation with Cd2+ reinforced culture medium. Intracellular free Cd2+ cation and Cd-MTs are identified, along with Cd2+ transformation to Cd-glutathione (GSH) adduct/complex, as further demonstrated by ESI-MS.

Biosynthesis of CdS Quantum Dots Mediated by Volatile Sulfur Compounds Released by Antarctic Pseudomonas fragi.

Previously we reported the biosynthesis of intracellular cadmium sulfide quantum dots (CdS QDs) at low temperatures by the Antarctic strain Pseudomonas fragi GC01. Here we studied the role of volatile sulfur compounds (VSCs) in the biosynthesis of CdS QDs by P. fragi GC01. The biosynthesis of nanoparticles was evaluated in the presence of sulfate, sulfite, thiosulfate, sulfide, cysteine and methionine as sole sulfur sources. Intracellular biosynthesis occurred with all sulfur sources tested. However, extracellular biosynthesis was observed only in cultures amended with cysteine (Cys) and methionine (Met). Extracellular nanoparticles were characterized by dynamic light scattering, absorption and emission spectra, energy dispersive X-ray, atomic force microscopy, transmission electron microscopy, X-ray diffraction and X-ray photoelectron spectroscopy. Purified QDs correspond to cubic nanocrystals of CdS with sizes between 2 and 16 nm. The analysis of VSCs revealed that P. fragi GC01 produced hydrogen sulfide (H2S), methanethiol (MeSH) and dimethyl sulfide (DMS) in the presence of sulfate, Met or Cys. Dimethyl disulfide (DMDS) was only detected in the presence of Met. Interestingly, MeSH was the main VSC produced in this condition. In addition, MeSH was the only VSC for which the concentration decreased in the presence of cadmium (Cd) of all the sulfur sources tested, suggesting that this gas interacts with Cd to form nanoparticles. The role of MeSH and DMS on Cds QDs biosynthesis was evaluated in two mutants of the Antarctic strain Pseudomonas deceptionensis M1T: megL– (unable to produce MeSH from Met) and mddA– (unable to generate DMS from MeSH). No biosynthesis of QDs was observed in the megL– strain, confirming the importance of MeSH in QD biosynthesis. In addition, the production of QDs in the mddA– strain was not affected, indicating that DMS is not a substrate for the biosynthesis of nanoparticles. Here, we confirm a link between MeSH production and CdS QDs biosynthesis when Met is used as sole sulfur source. This work represents the first report that directly associates the production of MeSH with the bacterial synthesis of QDs, thus revealing the importance of different VSCs in the biological generation of metal sulfide nanostructures.

The human ABCB6 protein is the functional homologue of HMT-1 proteins mediating cadmium detoxification

ABCB6 belongs to the family of ATP-binding cassette (ABC) transporters, which transport various molecules across extra- and intra-cellular membranes, bearing significant impact on human disease and pharmacology. Although mutations in the ABCB6 gene have been linked to a variety of pathophysiological conditions ranging from transfusion incompatibility to pigmentation defects, its precise cellular localization and function is not understood. In particular, the intracellular localization of ABCB6 has been a matter of debate, with conflicting reports suggesting mitochondrial or endolysosomal expression. ABCB6 shows significant sequence identity to HMT-1 (heavy metal tolerance factor 1) proteins, whose evolutionarily conserved role is to confer tolerance to heavy metals through the intracellular sequestration of metal complexes. Here, we show that the cadmium-sensitive phenotype of Schizosaccharomyces pombe and Caenorhabditis elegans strains defective for HMT-1 is rescued by the human ABCB6 protein. Overexpression of ABCB6 conferred tolerance to cadmium and As(III) (As2O3), but not to As(V) (Na2HAsO4), Sb(V), Hg(II), or Zn(II). Inactivating mutations of ABCB6 abolished vacuolar sequestration of cadmium, effectively suppressing the cadmium tolerance phenotype. Modulation of ABCB6 expression levels in human glioblastoma cells resulted in a concomitant change in cadmium sensitivity. Our findings reveal ABCB6 as a functional homologue of the HMT-1 proteins, linking endolysosomal ABCB6 to the highly conserved mechanism of intracellular cadmium detoxification.

Bioaccumulation and distribution of cadmium by Burkholderia cepacia GYP1 under oligotrophic condition and mechanism analysis at proteome level

Bacteria have been applied for the bioremediation of cadmium-contaminated environment. Less is known about the bioaccumulation of high concentration of Cd over time under the oligotrophic environment. Burkholderia cepacia GYP1, which was isolated from multiple heavy metal contaminated farmland, was studied for its bioaccumulation mechanism of Cd under oligotrophic condition. GYP1 possessed highly accumulation capacity for cadmium reaching 116 mg Cd/g biomass (dry weight). ATR-FTIR, electron microscopy, flow cytometry along with subcellular fraction demonstrated that the uptake and distribution of cadmium varied with the increased amount of cadmium of GYP1 cell during the 7-day treatment time: the accumulation of cadmium was mainly on the outer membrane at the beginning (within 1 day), and the intracellular cadmium kept increased and held stable after 2 days, after that, the increased amount of cadmium mainly located extracellularly, related to the secreted EPS. Further mechanism analysis of bioaccumulation of Cd by GYP1 based on iTRAQ-based proteomics showed that Cd(II) could trigger the up-regulation of the Cd2+/Zn2+-exporting ATPase, type VI protein secretion systems, and glutathione-S-transferase that are related to cadmium response, which may contribute to maintain the intracellular cadmium homeostasis. In summary, the immobilization of Cd(II) by B. cepacia GYP1 contains three steps:(1) fast immobilization of Cd(II) on the cell surface coordinated with functional groups, (2) transport of Cd(II) to cells and accumulation in cytoplasm, and (3) efflux of intracellular Cd(II) depended on energy and the entrapped or adsorbed of extracellular Cd(II) by EPS. Our study provided the understanding of the cadmium accumulation process of B. cepacia GYP1 under oligotrophic condition, which would be helpful in bioremediation of natural cadmium contaminated environment.

Time-dependent response of A549 cells upon exposure to cadmium.

Cadmium is considered one of the most harmful carcinogenic heavy metals in the human body. Although many scientists have performed research on cadmium toxicity mechanism, the toxicokinetic process of cadmium toxicity remains unclear. In the present study, the kinetic response of proteome in/and A549 cells to exposure of exogenous cadmium was profiled. A549 cells were treated with cadmium sulfate (CdSO4 ) for different periods and expressions of proteins in cells were detected by two-dimensional gel electrophoresis. The kinetic expressions of proteins related to cadmium toxicity were further investigated by reverse transcription-polymerase chain reaction and western blotting. Intracellular cadmium accumulation and content fluctuation of several essential metals were observed after 0-24hours of exposure by inductively coupled plasma mass spectrometry. Fifty-four protein spots showed significantly differential responses to CdSO4 exposure at both 4.5 and 24hours. From these proteins, four expression patterns were concluded. Their expressions always exhibited a maximum abundance ratio after CdSO4 exposure for 24hours. The expression of metallothionein-1 and ZIP-8, concentration of total protein, and contents of cadmium, zinc, copper, cobalt and manganese in cells also showed regular change. In synthesis, the replacement of the essential metals, the inhibition of the expression of metal storing protein and the activation of metal efflux system are involved in cadmium toxicity.

Cadmium detoxification induced by salt stress improves cadmium tolerance of multi-stress-tolerant Pichia kudriavzevii

Heavy metal tolerance of microorganisms is the basis of heavy metal removal by growing cells. In this study, a cross-protection effect generated by salt stress significantly enhanced the cadmium tolerance of multi-stress-tolerant Pichia kudriavzevii. Comparative transcriptome analysis using RNA-Seq linked with physiological and biochemical observation was used to elucidate the underlying mechanisms of the improved cadmium tolerance. The expression of cadmium transport related genes (GSTY2, GLR1, GLO2, YCF1 and YOR1), GSH content and GST activity were elevated by salt stress, suggesting enhanced cadmium conjugation and detoxification in yeast cells. The inhibited cadmium uptake by ZRT1 and enhanced cadmium efflux by YOR1 contributed to the decrease in the intracellular cadmium concentration. The improved expression of antioxidant enzyme genes (SOD1, SOD2, SOD6, CAT1 and PRXIID), along with the enhanced activities of antioxidant enzymes (SOD, CAT and POD) resulted in a decrease in cadmium-induced ROS production, protein carbonylation, lipid peroxidation and cell death. The abundant expression of heat shock protein genes (HSP12, HSP10 and SSC1) and genes related to trehalose synthesis (TPS1 and TSL1) induced by salt stress protected yeast cells against complex stress conditions, contributing to the improved cadmium tolerance. These findings will be useful to develop cadmium-tolerant yeasts for cadmium removal by growing cells.

Bioaccumulation of cadmium by Enterobacter sp. and enhancement of rice seedling growth under cadmium stress

Bacteria-mediated plant growth promotion and bioremediation of heavy metal containing soil is a widely accepted eco-friendly method. The present study is aimed to screen out cadmium resistant bacterial strain from metal contaminated rice rhizosphere and evaluate its effects on the growth of rice seedlings under cadmium stress. Among four different isolates (designated as S1, S2, S3 and S5), the S2 isolate was screened on the basis of different PGP traits and multi heavy metal resistance (minimum inhibitory concentration for cadmium, lead and arsenic were 3500, 2500 and 1050 µg/ml respectively). The selected S2 strain has ability to produce ACC deaminase (236.11 ng α-keto-butyrate/mg protein/h), IAA (726 µg/ml), solubilize phosphate (73.56 ppm) and fix nitrogen (4.4 µg of nitrogen fixed/h/mg protein). The selected strain was identified as Enterobacter sp. on the basis of phenotypic characterization, MALDI-TOF MS analysis of ribosomal proteins, FAME analysis and 16 S rDNA sequence homology. The high cadmium removal efficiency (> 95%) of this strain from the growth medium was measured by Atomic Absorption Spectrophotometer and it was due to intracellular cadmium accumulation evidenced by SEM-EDX-TEM-EDX study. SEM analysis also revealed no distortion of surface morphology of this strain even grown in the presence of high cadmium concentration (3000 µg/ml). Inoculation of this strain with rice seedlings significantly enhanced various morphological, biochemical characters of seedling growth compared with un-inoculated seedlings under Cd stress. The strain also exhibited alleviation of cadmium-induced oxidative stress, reduction of stress ethylene and decreased the accumulation of cadmium in seedlings as well that conferred cadmium tolerance to the plant. Thus the S2 strain could be considered as a potent heavy metal resistant PGPR applicable in heavy metal contaminated agricultural soil for bioremediation and plant growth promotion as well. Main findingA cadmium resistant plant growth promoting Enterobacter sp. was isolated that accumulated cadmium evidenced by SEM-TEM-EDX study. It reduced Cd uptake and enhanced growth in rice seedlings.

Engineered Protein Nanopore for Real-time Monitoring Single-molecule Reaction Between Cadmium Ion and Glutathione

Abstract The complexation of glutathione (GSH) with divalent cadmium ion has been used as a typical model for investigating the coordination chemistry of sulfydryl-containing peptides and heavy metal ions, which is essential to understand the mechanism of intracellular cadmium detoxification. In this study, the single-molecule reaction between GSH molecule and Cd2+ ion was monitored in real time by a nanoreactor that formed by a mutant (M113R)7 α-hemolysin (αHL) protein nanopore equipped with a novel per-6-quaternary ammonium-β-cyclodextrin (p-QABCD). The reaction pathways, intermediates, and products could be recognized by analyzing the current fluctuations. The reaction between Cd2+ and GSH was highly dependent on solution pH value. Cd(GSH)2 was the only final product at pH 7.4, while both Cd(GSH)2 and Cd2(GSH)2 were present at pH 9.0. The formation of Cd2(GSH)2 follows two possible pathways: (1) one Cd2+ ion first coordinates with the thiol group of two GSH molecules to form Cd(GSH)2, and then the second Cd2+ ion quickly incorporates with the deprotonated amino group of Cd(GSH)2 to produce Cd2(GSH)2; (2) two Cd2+ ions separately coordinate with the thiol and deprotonated amino group of one GSH molecule to yield Cd2(GSH)1, and the second GSH molecule binds Cd2+ ions quickly to form Cd2(GSH)2. The free-labeling and free-modifying method for monitoring single-molecule chemical reaction was simple and sensitive, which would be important to further understand intracellular mechanisms of detoxification of heavy metals. This work greatly expands the research field of single-molecule nanopore technique.

Cadmium-induced IL-6 and IL-8 expression and release from astrocytes are mediated by MAPK and NF-κB pathways

Chronic exposure to cadmium has been linked to brain cancers, learning disabilities and memory deficits. Previous studies of cadmium toxicity in the central nervous system report cadmium induces oxidative stress in neurons and astrocytes. In the peripheral system, cadmium promotes interleukin-6 (IL-6) and IL-8 production and release. Elevation of IL-6 expression is linked to the pathogenesis of neurodegenerative diseases and astrogliosis. IL-8 plays a role in angiogenesis of gliomas and neurodegenerative diseases. Herein, the effects of non-toxic concentrations of cadmium on the production of IL-6 and IL-8 and the underlying mechanisms were investigated. U-87 MG human astrocytoma cells and primary human astrocytes were exposed to cadmium chloride. At 24h post-exposure to 1 and 10μM, levels of intracellular cadmium in U-87 MG cells were 11.89±3.59 and 53.08±7.59μg/g wet weight, respectively. These concentrations had minimal effects on cell morphology and viability. IL-6 and IL-8 mRNA levels and secretion increased in dose- and time-dependent manners post cadmium exposure. Acute exposure to cadmium increased phosphorylation of ERK1/2, p38 MAPK, and p65 NF-κB. Pretreatment with U0126–an inhibitor of MEK1 and MEK2 kinases–SB203580–a p38 MAPK inhibitor–and SC-514–an IKKβ inhibitor–suppressed cadmium-induced IL-8 expression and release. Upregulation of cadmium-induced IL-6 was inhibited by U0126 and SC-514, but not SB203580. On the other hand, SP600125–a JNK inhibitor–and celecoxib–a selective COX-2 inhibitor–had no effect on production of both cytokines. In conclusion, non-toxic concentrations of cadmium can stimulate IL-6 and IL-8 release through MAPK phosphorylation and NF-κB activation. Suppressing IL-6 and IL-8 production could be novel approaches to prevent cadmium-induced angiogenesis in gliomas and inflammation in the brain.

Induction of cytotoxic and genotoxic damage following exposure of V79 cells to cadmium chloride

Cadmium is a toxic heavy metal with many industrial and commercial uses resulting in widespread environmental pollution. Exposure to cadmium occurs occupationally, through tainted food, tobacco leaves, and the environment. Cadmium exposure has been linked to neurological and respiratory problems and cancer. Past studies have examined cytotoxicity, DNA damage, micronuclei formation, or mutagenesis individually, but no study has examined these endpoints together and correlated them with intracellular cadmium concentrations. This study examines the cytotoxic, genotoxic, and mutagenic effects of cadmium exposure in V79 cells providing a correlation between the induction of DNA damage, the generation of mutations, and intracellular cadmium concentration. Cells exposed to 2.5–40μM cadmium exhibited a concentration-dependent decrease in survival which correlated with increases in PARP cleavage and caspase activity. Cytotoxicity was detected at intracellular cadmium concentrations of 12.9ppb. Exposure to cadmium also resulted in the production of single and double strand breaks and the induction micronuclei and mutations in the Hprt gene. Collectively, this study demonstrates that exposure of V79 cells to cadmium results in the induction of apoptosis which is related to the formation of DNA strand breaks and genotoxic damage.

A blood pressure-associated variant of theSLC39A8gene influences cellular cadmium accumulation and toxicity

Genome-wide association studies have revealed a relationship between inter-individual variation in blood pressure and the single nucleotide polymorphism rs13107325 in the SLC39A8 gene. This gene encodes the ZIP8 protein which co-transports divalent metal cations, including heavy metal cadmium, the accumulation of which has been associated with increased blood pressure. The polymorphism results in two variants of ZIP8 with either an alanine (Ala) or a threonine (Thr) at residue 391. We investigated the functional impact of this variant on protein conformation, cadmium transport, activation of signalling pathways and cell viability in relation to blood pressure regulation. Following incubation with cadmium, higher intracellular cadmium was detected in cultured human embryonic kidney cells (HEK293) expressing heterologous ZIP8-Ala391, compared with HEK293 cells expressing heterologous ZIP8-Thr391. This Ala391-associated cadmium accumulation also increased the phosphorylation of the signal transduction molecule ERK2, activation of the transcription factor NFκB, and reduced cell viability. Similarly, vascular endothelial cells with the Ala/Ala genotype had higher intracellular cadmium concentration and lower cell viability than their Ala/Thr counterpart following cadmium exposure. These results indicate that the ZIP8 Ala391-to-Thr391 substitution has an effect on intracellular cadmium accumulation and cell toxicity, providing a potential mechanistic explanation for the association of this genetic variant with blood pressure.

Fluorescence detection of intracellular cadmium with Leadmium Green.

Leadmium Green is a commercially available, small molecule, fluorescent probe advertised as a detector of free intracellular cadmium (Cd(2+)) and lead (Pb(2+)). Leadmium Green has been used in various paradigms, such as tracking Cd(2+) sequestration in plant cells, heavy metal export in protozoa, and Pb(2+) absorption by vascular endothelial cells. However very little information is available regarding its affinity and selectivity for Cd(2+), Pb(2+), and other metals. We evaluated the in vitro selectivity of Leadmium Green using spectrofluorimetry. Consistent with manufacturer's claims, Leadmium Green was sensitive to Cd(2+) (KD~600nM) and also Pb(2+) (KD~9.0nM) in a concentration-dependent manner, and furthermore proved insensitive to Ca(2+), Co(2+), Mn(2+) and Ni(2+). Leadmium Green also responded to Zn(2+) with a KD of ~82nM. Using fluorescence microscopy, we evaluated Leadmium Green in live mouse hippocampal HT22 cells. We demonstrated that Leadmium Green detected ionophore-mediated acute elevations of Cd(2+) or Zn(2+) in a concentration-dependent manner. However, the maximum fluorescence produced by ionophore-delivered Zn(2+) was much less than that produced by Cd(2+). When tested in a model of oxidant-induced liberation of endogenous Zn(2+), Leadmium Green responded weakly. We conclude that Leadmium Green is an effective probe for monitoring intracellular Cd(2+), particularly in models where Cd(2+) accumulates rapidly, and when concomitant fluctuations of intracellular Zn(2+) are minimal.

A Model of Cadmium Uptake and Transport in Caco-2 Cells.

We created a physiologically substantiated kinetic model of cadmium transport and toxicity in intestinal cell model (Caco-2 cells). Transcriptome profiling of Caco-2 cells revealed high content of transporter DMT1 and ZIP14 and intensive expression of some calcium channels of the CACN family. The mathematical model describing three types of transporters, as well as intracellular cadmium binding with metallothionein and excretion through the basolateral membrane allowed us to construct cadmium uptake and transport curves that approximated the previously obtained experimental data. Using the proposed model, we determined toxic intracellular cadmium concentration leading to cell death and impairing the integrity of cell monolayer and described cadmium transport in this case.

Characterization of Leadmium Green for Fluorescence Detection of Intracellular Cadmium

Cadmium is a common industrial pollutant, but the study of cadmium cytotoxicity is difficult in part due to the absence of a probe that can detect cadmium in living cells without the confounds of contaminating metals. Leadmium Green (LG) is a commercially available, small molecule, fluorescent probe advertised as a detector of intracellular cadmium and lead. LG has been used to monitor cadmium sequestration in plant cells, heavy metal export in a protozoan, and lead absorption by vascular endothelial cells. However very little information is available regarding its affinity and selectivity for cadmium, lead, and other metals. Using a cell‐impermeant form, we evaluated the in vitro selectivity of LG using spectrofluorometry. Consistent with manufacturer's claims, LG responded robustly to cadmium (KD 603nM) and also lead (KD 8.65nM) in a concentration‐dependent manner, and furthermore proved insensitive to calcium, cobalt, manganese and nickel. However, LG also responded to zinc with a KD of 81.9nM. Using fluorescence microscopy, we evaluated the utility of LG in live mouse hippocampal HT22 cells. We demonstrated that LG detects ionophore‐mediated elevations of cadmium or zinc in a concentration‐dependent manner. In comparison to zinc changes reported by the widely used zinc fluorescent probe FluoZin‐3, LG yielded a relatively small dynamic range. In conclusion, LG may have utility as an intracellular cadmium probe, however data must be interpreted cautiously due to potential contribution of zinc to the signal.

Cadmium-induced oxidative stress, response of antioxidants and detection of intracellular cadmium in organs of moso bamboo (Phyllostachys pubescens) seedlings

Moso bamboo (Phyllostachys pubescens (Pradelle) Mazel ex J.Houz.) is recognized as a potential phytoremediation plant due to its huge biomass and high tolerance to environmental stresses. The objectives of this study were to investigate mechanism related to cadmium (Cd) tolerance and to evaluate Cd accumulation capacity of moso bamboo. The results of the pot experiment showed that Cd accumulation by bamboo increased with increasing the Cd levels in soil and the values in stem ranged from 28.51 to 132.13 mg kg−1. Meanwhile chlorophyll in leaves and total biomass showed a decreasing trend. The bioaccumulation factors (BAF) for roots and stem in all the treatments were more than 1.0 and the translocation factor (TF) ranged from 0.70 to 1.06. In hydroponics experiment, the concentrations of malondialdehyde (MDA) in the leaves were significantly increased in Cd treated plants as compared with control. The activities of superoxide dismutase (SOD) and peroxidase (POD) were enhanced at initial stage and then decreased consistently with the increase of Cd addition. The proline concentrations were also increased due to the presence of Cd, particularly at 25 μM Cd treatment. According to TEM-EDX analysis, the cytoplasm was the main site for accumulation of Cd in moso bamboo. On the basis of overall results, it is suggested that moso bamboo could be successfully used for the remediation of low Cd (no more than 5 mg kg−1) contaminated soils.

Modulation of cadmium bioaccumulation and enhancing cadmium tolerance in Pichia kudriavzevii by sodium chloride preincubation.

Application of growing microorganisms for cadmium removal is limited by the sensitivity of living cells to cadmium. The effects of sodium chloride (NaCl) preincubation on the cadmium bioaccumulation and tolerance of Pichia kudriavzevii and Saccharomyces cerevisiae were investigated in this study. NaCl preincubation significantly reduced the intracellular and cell-surface cadmium bioaccumulation of P. kudriavzevii at both 6 and 20 mg L(-1) cadmium, while no obvious effect was observed in S. cerevisiae except that the intracellular cadmium bioaccumulation at 20 mg L(-1) cadmium was reduced obviously by 20-60 g L(-1) NaCl. For both yeasts, the improved contents of protein and proline after NaCl preincubation contributed to the cadmium tolerance. The thiol contents in P. kudriavzevii under cadmium stress were alleviated by NaCl preincubation, which might be due to the decrease of intracellular cadmium bioaccumulation. NaCl preincubation enhanced the contents of glycerol and trehalose in P. kudriavzevii under cadmium stress, while no acceleration was observed in S. cerevisiae. The results suggested that NaCl preincubation could be applied in cadmium removal by growing P. kudriavzevii to increase the cadmium tolerance of the yeast.