Exogenous Abscisic Acid Alleviates Cadmium Toxicity by Restricting Cd2+ Influx in Populus euphratica Cells

Abscisic acid (ABA), a widely known phytohormone involved in the plant response to abiotic stress, plays a vital role in mitigating Cd²⁺ toxicity in herbaceous species. However, the role of ABA in ameliorating Cd²⁺ toxicity in woody species is largely unknown. In the present study, we investigated ABA restriction on Cd²⁺ uptake and the relevance to Cd²⁺ stress alleviation in Cd²⁺-hypersensitive Populus euphratica. ABA (5 μM) markedly improved cell viability and growth but reduced membrane permeability in CdCl₂ (100 μM)-stressed P. euphratica cells. Moreover, ABA significantly increased the activity of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), and ascorbate peroxidase (APX), contributing to the scavenging of Cd²⁺-elicited H₂O₂ within P. euphratica cells during the period of CdCl₂ exposure (100 μM, 24–72 h). ABA alleviation of Cd²⁺ toxicity was mainly the result of ABA restriction of Cd²⁺ uptake under Cd²⁺ stress. Steady-state and transient flux recordings showed that ABA inhibited Cd²⁺ entry into Cd²⁺-shocked (100 μM, 30 min) and short-term-stressed P. euphratica cells (100 μM, 24–72 h). Non-invasive micro-test technique data showed that H₂O₂ (3 mM) stimulated the Cd²⁺-elicited Cd²⁺ influx but that the plasma membrane (PM) Ca²⁺ channel inhibitor LaCl₃ blocked it, suggesting that the Cd²⁺ influx was through PM Ca²⁺-permeable channels. These results suggested that ABA up-regulated antioxidant enzyme activity in Cd²⁺-stressed P. euphratica and that these enzymes scavenged the Cd²⁺-elicited H₂O₂ within cells. The entry of Cd²⁺ through the H₂O₂-mediated Ca²⁺-permeable channels was subsequently restricted; thus, Cd²⁺ buildup and toxicity were reduced in the Cd²⁺-hypersensitive species, P. euphratica.

     

Cadmium (Cd2+) disrupts E-cadherin-dependent cell-cell junctions in MDCK cells

Summary Previous studies from our laboratory have shown that Cd²⁺ can selectively disrupt E-cadherin-dependent cell-cell junctions in the porcine renal epithelial cell line, LLC-PK₁. The objective of the present studies was to determine whether or not Cd²⁺ could produce similar effects in Madin-Darby canine kidney (MDCK) cells, an immortal epithelial cell line derived from dog kidney. This is an important issue because MDCK cells have been used extensively as a model system to study the basic mechanisms of E-cadherin-dependent cell-cell adhesion. MDCK cells on permeable membrane supports were exposed to Cd²⁺ by adding CdCl₂ to either the apical or the basolateral compartment. The integrity of cell-cell junctions was assessed by morphologic observation of the cells and by monitoring the transepithelial electrical resistance. The results showed that exposure to 10–40 μM Cd²⁺ for 15 min-4 h caused the cells to separate from each other without detaching from the growing surface. The separation of the cells was accompanied by a marked drop in the transepithelial electrical resistance, a loss of E-cadherin from the cell-cell contacts, and a reorganization of the actin cytoskeleton. These effects were much more pronounced when Cd²⁺ was added basolaterally than when it was added apically. Moreover, the effects of Cd²⁺ were qualitatively similar to those observed when the cells were incubated in Ca²⁺-free medium. These results show that Cd²⁺ can disrupt E-cadherin-dependent cell-cell junctions in MDCK cells, and they indicate that this cell line would be an appropriate model for further mechanistic studies in this area.     

Endoplasmic reticulum is a major target of cadmium toxicity in yeast

Cadmium (Cd²⁺) is a very toxic metal that causes DNA damage, oxidative stress and apoptosis. Despite many studies, the cellular and molecular mechanisms underlying its high toxicity are not clearly understood. We show here that very low doses of Cd²⁺ cause ER stress in Saccharomyces cerevisiae as evidenced by the induction of the unfolded protein response (UPR) and the splicing of HAC1 mRNA. Furthermore, mutant strains (Δire1 and Δhac1) unable to induce the UPR are hypersensitive to Cd²⁺, but not to arsenite and mercury. The full functionality of the pathways involved in ER stress response is required for Cd²⁺ tolerance. The data also suggest that Cd²⁺‐induced ER stress and Cd²⁺ toxicity are a direct consequence of Cd²⁺ accumulation in the ER. Cd²⁺ does not inhibit disulfide bond formation but perturbs calcium metabolism. In particular, Cd²⁺ activates the calcium channel Cch1/Mid1, which also contributes to Cd²⁺ entry into the cell. The results reinforce the interest of using yeast as a cellular model to study toxicity mechanisms in eukaryotic cells.     

Catch me if you can! Novel aspects of cadmium transport in mammalian cells

Cadmium (Cd²⁺) is a nonessential divalent metal ion that causes toxicity in multiple organs in humans. In order for toxicity to occur Cd²⁺ must first enter cells by utilizing transport pathways for essential metals. This review focuses on studies in which Cd²⁺ transport was directly demonstrated by electrophysiological, radiotracer or Cd²⁺-sensitive fluorescent dye techniques. The chemistry of Cd²⁺ and metal ions in general is addressed in the context of properties relevant for transport through membrane proteins, such as hydration energy. Apart from transport by the ZIP transporters SLC39A8 and SLC39A14, which is not topic of the review, uptake of free Cd²⁺ has been demonstrated for the Fe²⁺/H⁺ cotransporter divalent metal transporter 1. Moreover, the multiligand endocytic receptors megalin and cubilin take up cadmium-metallothionein complexes via receptor-mediated endocytosis. The role of ATP binding cassette transporters in Cd²⁺ efflux from cells is also discussed. Both the multidrug resistance-associated protein 1 and cystic fibrosis transmembrane conductance regulator are likely to transport cadmium–glutathione complexes out of cells, whereas transport of free Cd²⁺ by the multidrug resistance P-glycoprotein remains controversial. Finally, arguments for and against Cd²⁺ transport by Ca²⁺ channels are presented. Most N- and L-type Ca²⁺ channels are closed at resting membrane potential (with the exception of CaV1.3 channels) and therefore unlikely to allow significant Cd²⁺ influx under physiological conditions. CaV3.1 and CaV3.2 T-type calcium channels are permeated by divalent metal ions, such as Fe²⁺ and Mn²⁺ because of considerable “window” currents close to resting membrane potential and could be responsible for tonic Cd²⁺ entry. TRPM7 and the mitochondrial Ca²⁺ uniporter are other likely candidates for Cd²⁺ transporters, whereas the role of Orai proteins, the store-operated calcium channels carrying Ca²⁺ release-activated Ca²⁺ current, in Cd²⁺ influx remains to be investigated.     

Development of a broad-spectrum fluorescent heavy metal bacterial biosensor

Bacterial biosensors can measure pollution in terms of their actual toxicity to living organisms. A recombinant bacterial biosensor has been constructed that is known to respond to toxic levels of Zn²⁺, Cd²⁺ and Hg²⁺. The zinc regulatory gene zntR and zntA promoter from znt operon of E. coli have been used to trigger the expression of GFP reporter protein at toxic levels of these ions. The sensor was induced with 3–800?ppm of Zn²⁺, 0.005–4?ppm of Cd²⁺ and 0.001–0.12?ppm of Hg²⁺ ions. Induction studies were also performed in liquid media to quantify GFP fluorescence using fluorimeter. To determine the optimum culture conditions three different incubation periods (16, 20 and 24?h) were followed. Results showed an increased and consistent fluorescence in cells incubated for 16?h. Maximum induction for Zn²⁺, Cd²⁺ and Hg²⁺ was observed at 20, 0.005 and 0.002?ppm, respectively. The pPROBE-zntR-zntA biosensor reported here can be employed as a primary screening technique for aquatic heavy metal pollution.     

Heavy metal uptake by intact cells and protoplasts of Aureobasidium pullulans

Protoplasts prepared from yeast-like cells, hyphae and chlamydospores of Aureobasidium pullulans can take up heavy metals such as Zn²⁺, Co²⁺, Cd²⁺ and Cu²⁺. In relation to intact cells, the sensitivity of protoplasts to Cu²⁺ and Cd²⁺ was increased although chlamydospore protoplasts were more tolerant than yeast-like cell protoplasts. Surface binding of metals was reduced in protoplasts as compared with intact cells and this reduction was particularly evident for chlamydospore protoplasts. At the highest concentrations used, uptake of Zn²⁺, Co²⁺ and Cd²⁺ by yeast-like cell protoplasts was greater than that observed in intact cells which may have been due to toxicity, especially for Cd²⁺, resulting in increased membrane permeability, though for Zn²⁺ and Co²⁺ some barrier effect of the cell wall could not be completely discounted. Chlamydospore protoplasts were capable of intracellular metal uptake, unlike intact chlamydospores, and for Zn²⁺, uptake appeared to be via a different system less specific than that of the other cell types. For chlamydospores, the use of protoplasts confirmed the importance of the cell wall in preventing entry of metal ions into the cell.     

Spectroscopic and Microscopic Studies on the Mechanisms of Mitochondrial Toxicity Induced by Different Concentrations of Cadmium

The deleterious action of Cd²⁺ on rat liver mitochondria was investigated in this work using spectroscopic and microscopic methods. The concentration dependence of Cd²⁺ on mitochondrial swelling, membrane potential and membrane fluidity was studied. Our aim was to detect the active sites of Cd²⁺ in the mitochondrial membrane treatments with cyclosporin A (CsA) and EGTA on the mitochondrial permeability transition (MPT) induced by low and high concentrations of Cd²⁺. The protective effects of dithiothreitol, human serum albumin and monobromobimane⁺ on Cd²⁺-induced MPT were also monitored. All of these investigations indicated that Cd²⁺ can directly affect MPT at two separate localization sites at different concentrations: the classic Ca²⁺ triggering site and the thiol (–SH) groups of membrane proteins matched by MPT pore opening (defined as “S” site). At the high concentration of Cd²⁺, other free –SH groups in the mitochondrial matrix may be involved in this process. These findings were supported by transmission electron microscopy and shed light on the toxic mechanism of Cd²⁺ on mitochondria.     

Cadmium and transport of ions and substances across cell membranes and epithelia

Toxic metals such as cadmium (Cd²⁺) pose serious risks to human health. However, even though the importance of Cd²⁺ as environmental health hazards is now widely appreciated, the specific mechanisms by which it produces its adverse effects have yet to be fully elucidated. Cd²⁺ is known to enter cells, it binds and interacts with a multitude of molecules, it may indirectly induce oxidative stress and interfere with gene expression and repair of DNA. It also interacts with transport across cell membranes and epithelia and may therefore disturb the cell’s homeostasis and function. Interaction with epithelial transport, especially in the kidney and the liver, may have serious consequences in general health. A lot of research still needs to be done to understand the exact way in which Cd²⁺ interferes with these transport phenomena. It is not always clear whether Cd²⁺ has primary or secondary effects on cell membrane transport. In the present review we try to summarize the work that has been done up to now and to critically discuss the relevance of the experimental work in vitro with respect to the in vivo situation.     

Characterization of the Human Intestinal Calcium Transporter, CaT1, Stably Expressed in CHO Cells

The human calcium transporter, hCaT1, was cloned and analyzed. The obtained amino acid sequence was slightly different from the ortholog of hCaT1 which had been identified by Peng et al. (2000. Biochem. Biophys. Res. Commun 278: 326-332). An mRNA analysis of human gastrointestinal segments demonstrates that hCaT1 was expressed in the stomach, duodenum, jejunum, ileum, ileocecum, cecum, ascending colon, transverse colon, descending colon, and, at very low levels, in the esophagus and rectum. hCaT1 was transiently expressed by transfecting COS-1 cells and was stably expressed by the transfected CHO cells. The transfected cells expressed hCaT1 with a molecular mass of 75 kDa. Stable expression of hCaT1 in the CHO cells increased the cellular uptake of Ca²⁺. hCaT1 was inhibited by La³⁺, Gd³⁺ and Cd²⁺, whereas Co²⁺, Fe²⁺, Mn²⁺ and Mg²⁺ showed no significant effects on the activity. Acidification of the extracellular solution to pH 5.5 reduced the ⁴⁵Ca²⁺uptake by hCaT1 in the CHO cells. The addition of lactose and raffinose had no effect on the ⁴⁵Ca²⁺ uptake, whereas galactose and glucose increased the ⁴⁵Ca²⁺ uptake. CHO cells stably expressing hCaT1 will be useful to detect and analyze food substances that could modulate the hCaT1 activity.     

The effect of cadmium on cell cycle control in suspension culture cells of soybean

Heavy metals inhibit plant growth. This proces may be directly or indirectly connected with mechanisms regulating cell division. We analyzed the effect of Cd²⁺ on cell cycle progression in partially synchronized soybean (Glycine max) cell suspension culture and followed the expression of cell cycle genes (cyclin B1 and cyclin-dependent kinase A - CDK-A). We have checked the hypothesis that Cd²⁺-induced impairment of cell division is connected with DNA damage. The [³H]-thymidine incorporation in cell cultures synchronized either with hydroxyurea (HU) or phosphate starvation have shown, that Cd²⁺ strongly affects the S phase of soybean cell cycle, by causing the earlier entry of cells into S phase and by decreasing the rate of DNA synthesis. RT-PCR analysis indicated that Cd²⁺ decreases the level of cyclin B1 mRNA and has no effect on CDK-A mRNA. The result of comet assay indicated the damaging effect of Cd²⁺ on DNA of soybean cells. We suggest that Cd²⁺ affects plant cell cycle at two major checkpoints: the G1/S — by damaging of DNA, and G2/M - by decreasing the level of cyclin B1 mRNA     

Rat kidney epithelial cell culture for metal toxicity studies

Summary Evaluation of the potential adverse human health effects of low-level chronic exposure to heavy metals is dependent on the basic knowledge of the cellular and molecular toxicology of these metals. The use of various cell culture systems has greatly facilitated our knowledge of the cellular effects. Inasmuch as most of the acute and chronic toxic effects of metals occur primarily on the renal proximal tubules, the development of a rat kidney epithelial cell culture has provided a unique system to study the uptake and mechanism of toxicity of metals and their intracellular binding ligands. In the presence ofd-valine, fibroblast growth was retarded and a primary epithelial monolayer culture was selectively grown from rat kidney cells. A distinct difference in the uptake of chemically similar divalent metals, such as Pb²⁺, Hg²⁺, Cd²⁺, and Zn²⁺, was observed in these cells. Both Pb²⁺ and Hg²⁺ were more avidly taken up by kidney cells than Cd²⁺ and Zn²⁺ salts and they also showed increased toxicity. On the other hand, the cellular uptake of Cd from cadmium-metallothionein (CdMT) was much less than from CdCl₂, but CdMT was about seven times more toxic than CdCl₂ when added to the renal cell culture. The cytotoxicity of CdCl₂ was decreased significantly with pretreatment of the cells with CdCl₂, although this had no effect on the toxicity of CdMT. The cellular toxicity of CdMT occurred probably during the process of its transport across the plasma membrane whereas that of CdCl₂ occurred after it had entered the cell. Thus rat kidney epithelial cells may be a useful tool to study the mechanism of renal toxicity of environmental chemicals and drugs. This work was funded by grants-in-aid of research from the Kidney Foundation of Canada.     

Cadmium inhibits mismatch repair by blocking the ATPase activity of the MSH2-MSH6 complex

Cadmium (Cd²⁺) is a known carcinogen that inactivates the DNA mismatch repair (MMR) pathway. In this study, we have tested the effect of Cd²⁺ exposure on the enzymatic activity of the mismatch binding complex MSH2–MSH6. Our results indicate that Cd²⁺ is highly inhibitory to the ATP binding and hydrolysis activities of MSH2–MSH6, and less inhibitory to its DNA mismatch binding activity. The inhibition of the ATPase activity appears to be dose and exposure time dependent. However, the inhibition of the ATPase activity by Cd²⁺ is prevented by cysteine and histidine, suggesting that these residues are essential for the ATPase activity and are targeted by Cd²⁺. A comparison of the mechanism of inhibition with N-ethyl maleimide, a sulfhydryl group inhibitor, indicates that this inhibition does not occur through direct inactivation of sulfhydryl groups. Zinc (Zn²⁺) does not overcome the direct inhibitory effect of Cd²⁺ on the MSH2–MSH6 ATPase activity in vitro. However, the increase in the mutator phenotype of yeast cells exposed to Cd²⁺ was prevented by excess Zn²⁺, probably by blocking the entry of Cd²⁺ into the cell. We conclude that the inhibition of MMR by Cd²⁺ is through the inactivation of the ATPase activity of the MSH2–MSH6 heterodimer, resulting in a dominant negative effect and causing a mutator phenotype.     

Nitric oxide promotes MPK6‐mediated caspase‐3‐like activation in cadmium‐induced Arabidopsis thaliana programmed cell death

Nitric oxide (NO), a vital cell‐signalling molecule, has been reported to regulate toxic metal responses in plants. This work investigated the effects of NO and the relationship between NO and mitogen‐activated protein kinase (MAPK) in Arabidopsis (Arabidopsis thaliana) programmed cell death (PCD) induced by cadmium (Cd²⁺) exposure. With fluorescence resonance energy transfer (FRET) analysis, caspase‐3‐like protease activation was detected after Cd²⁺ treatment. This was further confirmed with a caspase‐3 substrate assay. Cd²⁺‐induced caspase‐3‐like activity was inhibited in the presence of the NO‐specific scavenger 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (cPTIO), suggesting that NO mediated caspase‐3‐like protease activation under Cd²⁺ stress conditions. Pretreatment with cPTIO effectively inhibited Cd²⁺‐induced MAPK activation, indicating that NO also affected the MAPK pathway. Interestingly, Cd²⁺‐induced caspase‐3‐like activity was significantly suppressed in the mpk6 mutant, suggesting that MPK6 was required for caspase‐3‐like protease activation. To our knowledge, this is the first demonstration that NO promotes Cd²⁺‐induced Arabidopsis PCD by promoting MPK6‐mediated caspase‐3‐like activation.     

New Alternately Colored FRET Sensors for Simultaneous Monitoring of Zn2+ in Multiple Cellular Locations

Genetically encoded sensors based on fluorescence resonance energy transfer (FRET) are powerful tools for reporting on ions, molecules and biochemical reactions in living cells. Here we describe the development of new sensors for Zn²⁺based on alternate FRET-pairs that do not involve the traditional CFP and YFP. Zn²⁺ is an essential micronutrient and plays fundamental roles in cell biology. Consequently there is a pressing need for robust sensors to monitor Zn²⁺ levels and dynamics in cells with high spatial and temporal resolution. Here we develop a suite of sensors using alternate FRET pairs, including tSapphire/TagRFP, tSapphire/mKO, Clover/mRuby2, mOrange2/mCherry, and mOrange2/mKATE. These sensors were targeted to both the nucleus and cytosol and characterized and validated in living cells. Sensors based on the new FRET pair Clover/mRuby2 displayed a higher dynamic range and better signal-to-noise ratio than the remaining sensors tested and were optimal for monitoring changes in cytosolic and nuclear Zn²⁺. Using a green-red sensor targeted to the nucleus and cyan-yellow sensor targeted to either the ER, Golgi, or mitochondria, we were able to monitor Zn²⁺ uptake simultaneously in two compartments, revealing that nuclear Zn²⁺ rises quickly, whereas the ER, Golgi, and mitochondria all sequester Zn²⁺ more slowly and with a delay of 600–700 sec. Lastly, these studies provide the first glimpse of nuclear Zn²⁺ and reveal that nuclear Zn²⁺ is buffered at a higher level than cytosolic Zn²⁺.     

Rapid Recycling of Ca2+ between IP3-Sensitive Stores and Lysosomes

Inositol 1,4,5-trisphosphate (IP₃) evokes release of Ca²⁺ from the endoplasmic reticulum (ER), but the resulting Ca²⁺ signals are shaped by interactions with additional intracellular organelles. Bafilomycin A₁, which prevents lysosomal Ca²⁺ uptake by inhibiting H⁺ pumping into lysosomes, increased the amplitude of the initial Ca²⁺ signals evoked by carbachol in human embryonic kidney (HEK) cells. Carbachol alone and carbachol in combination with parathyroid hormone (PTH) evoke Ca²⁺ release from distinct IP₃-sensitive Ca²⁺ stores in HEK cells stably expressing human type 1 PTH receptors. Bafilomycin A₁ similarly exaggerated the Ca²⁺ signals evoked by carbachol or carbachol with PTH, indicating that Ca²⁺ released from distinct IP₃-sensitive Ca²⁺ stores is sequestered by lysosomes. The Ca²⁺ signals resulting from store-operated Ca²⁺ entry, whether evoked by thapsigargin or carbachol, were unaffected by bafilomycin A₁. Using Gd³⁺ (1 mM) to inhibit both Ca²⁺ entry and Ca²⁺ extrusion, HEK cells were repetitively stimulated with carbachol to assess the effectiveness of Ca²⁺ recycling to the ER after IP₃-evoked Ca²⁺ release. Blocking lysosomal Ca²⁺ uptake with bafilomycin A₁ increased the amplitude of each carbachol-evoked Ca²⁺ signal without affecting the rate of Ca²⁺ recycling to the ER. This suggests that Ca²⁺ accumulated by lysosomes is rapidly returned to the ER. We conclude that lysosomes rapidly, reversibly and selectively accumulate the Ca²⁺ released by IP₃ receptors residing within distinct Ca²⁺ stores, but not the Ca²⁺ entering cells via receptor-regulated, store-operated Ca²⁺ entry pathways.     

Cation channels in human embryonic kidney cells mediating calcium entry in response to extracellular low glucose

Glucose sensing mechanism has been intensively studied in pancreatic cells and neurons. Depolarization of membrane potential by closure of K_ATP , Kv and TASK channel, and subsequently Ca²⁺ entry via L-type voltage gated Ca²⁺ channel (VGCC) are implicated to mediate the signal transduction in these cells. However, the mechanism of non-excitable cells, which are lacking VGCC, for sensing glucose remains unclear. In this study, we utilized the calcium ratio measurement and patch clamping technique to study the effects of low glucose on [Ca²⁺]_i and currents in the human embryonic kidney epithelial cells (HEK 293). We found low glucose evoked a significant reversible [Ca²⁺]_i elevation in HEK 293 independent of the closure of Kv channels. This increase of [Ca²⁺]_i was mediated by Ca²⁺ entry across plasma membrane and exhibited a dosage dependent behaviour to external glucose concentration. The low glucose-induced entry of Ca²⁺ was characterized as a voltage independent behaviour and had cation permeability to Na⁺ and Ca²⁺. The modulation of PLC, AMPK, tyrosine kinase and cADPribose failed to regulate this glucose-sensitive Ca²⁺ entry. In addition, the entry of Ca²⁺ was insensitive to nifedipine, 2APB, SKF, La³⁺, Gd³⁺, and KBR9743, suggesting a novel signal pathway in mediating glucose sensing.     

Regulative mechanism of Ce3+ relieves DNA damage caused by Cd2+ in the kidney of silver crucian carp

The mechanism of Cd²⁺ on the DNA cleavage and Ce³⁺ on the DNA repair in the kidney of silver crucian carp (Carassius auratus gibelio) is investigated by agarose gel electrophoresis methods and assaying biochemical indexes. It proves that Cd²⁺ induces the classical laddering degradation of DNA in vivo, but DNA cleavage is repaired after injecting with a low Ce³⁺ concentration under various Cd²⁺ concentrations. The DNA cleavage caused by Cd²⁺ is the result of the activation of deoxyribonuclease (DNase) and accumulation of reactive oxygen species (ROS), and Cd²⁺ destroys the antioxidant system, which diminishes the activities of superoxide dismutase, catalase, and peroxidase, and the increase of the lipid peroxidation (LPO) level. However, Ce³⁺ could inhibit activation of Cd²⁺ on DNase activity, relieve inhibition of Cd²⁺ on activities of the antioxidant enzyme, and diminish ROS accumulation. The results show that Ce³⁺ could relieve the toxicity of Cd²⁺ to silver crucian carp.     

The involvement of HSP22 from bay scallop <Emphasis Type="Italic">Argopecten irradians</Emphasis> in response to heavy metal stress

Heat shock protein 22 (HSP22) is an important member of small heat shock protein (sHSP) subfamily which plays a key role in the process of protecting cells, facilitating the folding of nascent peptides, and responding to stress. In the present study, the cDNA of HSP22 was cloned from Argopecten irradians (designated as AiHSP22) by rapid amplification cDNA end (RACE) based on the expressed sequence tags (ESTs). The full-length cDNA of AiHSP22 was of 1,112 bp, with an open reading frame of 588 bp encoding a polypeptide of 195 amino acids. The deduced amino acid sequence of AiHSP22 showed high similarity to previously identified HSP22s. The expression patterns of AiHSP22 mRNA in different tissues and in haemocytes of scallops exposed to Cd²⁺, Pb²⁺ or Cu²⁺ were investigated by real-time quantitative RT-PCR. The mRNA of AiHSP22 was constitutively expressed in all examined tissues, including haemocyte, muscle, kidney, gonad, gill and heart. The expression level in heart and muscle was higher than that in other tissues. The mRNA level of AiHSP22 in haemocytes was up-regulated after a 10 days exposure of scallops to Cu²⁺, Pb²⁺ and Cd²⁺. However, the expression of AiHSP22 did not increase linearly along with the rise of heavy metal concentration. Different concentrations of the same metal resulted in different effects on AiHSP22 expression. The sensitive response of AiHSP22 to Cu²⁺, Pb²⁺ and Cd²⁺ stress indicated that it could be developed as an indicator of exposure to heavy metals for the pollution monitoring programs in aquatic environment.     

The role of metallothionein synthesis in cadmium- and zinc-resistant CHO-K1M cells

In order to probe the mechanism of survival at high concentrations of cadmium, a population of Cd²⁺-resistant Chinese hamster ovary cells (CHO-K1M), was obtained by selective pressure. This stable population of cells were characterized as tolerant to 200 μM Cd²⁺. In addition to the acquired resistance to Cd²⁺, the CHO-K1M cells also demonstrated resistance to 2 mM Zn²⁺. The CHO-K1M cells exhibit a diminished capacity to accumulate Cd²⁺ at low concentration (0.5 or 1.0 μM), which is not evident at high Cd²⁺ concentration. CHO-K1M cells demonstrated an induced synthesis of metallothionein as defined by physical characteristics and cysteine incorporation. The CHO-K1M cells cultured in the presence of 200 μM Cd²⁺ were determined to have an intracellular concentration of metallothionein representing more than 50-fold that observed in the wild-type cells. These results suggest that in CHO-K1M cells, the induction of metallothionein synthesis represents the important parameter involved in the determination of resistance to high levels of Cd²⁺ and Zn²⁺.