microM Cd Research Articles

Soft metal ions, Cd(II) and Hg(II), induce triple-stranded alpha-helical assembly and folding of a de novo designed peptide in their trigonal geometries.

We previously reported the de novo design of an amphiphilic peptide [YGG(IEKKIEA)4] that forms a native-like, parallel triple-stranded coiled coil. Starting from this peptide, we sought to regulate the assembly of the peptide by a metal ion. The replacement of the Ile18 and Ile22 residues with Ala and Cys residues, respectively, in the hydrophobic positions disrupted of the triple-stranded alpha-helix structure. The addition of Cd(II), however, resulted in the reconstitution of the triple-stranded alpha-helix bundle, as revealed by circular dichroism (CD) spectroscopy and sedimentation equilibrium analysis. By titration with metal ions and monitoring the change in the intensity of the CD spectra at 222 nm, the dissociation constant Kd was determined to be 1.5 +/- 0.8 microM for Cd(II). The triple-stranded complex formed by the 113Cd(II) ion showed a single 113Cd NMR resonance at 572 ppm whose chemical shift was not affected by the presence of Cl- ions. The 113Cd NMR resonance was connected with the betaH protons of the cysteine residue by 1H-113Cd heteronuclear multiple quantum correlation spectroscopy. These NMR results indicate that the three cysteine residues are coordinated to the cadmium ion in a trigonal-planar complex. Hg(II) also induced the assembly of the peptide into a triple-stranded alpha-helical bundle below the Hg(II)/peptide ratio of 1/3. With excess Hg(II), however, the alpha-helicity of the peptide was decreased, with the change of the Hg(II) coordination state from three to two. Combining this construct with other functional domains should facilitate the production of artificial proteins with functions controlled by metal ions.

Pattern of Stress Protein Expression in Human Lung Cell-Line A549 after Short- or Long-Term Exposure to Cadmium

Exposure to cadmium is associated with the development of pulmonary damage such as emphysema and lung cancer. This metal is also a powerful inducer of stress proteins in many biologic models. The present study was undertaken to evaluate whether an overexpression of the heat shock protein (hsp)72 stress protein, which indicates repair of damaged proteins, could be a sensitive and early biomarker of environmental pollution by Cd. In comparative studies, we examined the effects of exposure to Cd (as CdCl(2)) on the growth rate of the A549 pulmonary cell line, and (by Western blot analyses) on the induction of the hsp72 stress protein and metallothioneins (MTs). CdCl(2) exposure was studied for periods of 2 hr to 1 month. For short-term exposure (2-6 hr) to Cd concentrations higher than 50 microM, an overexpression of hsp72 appeared 6 hr later, suggesting that hsp72 might be considered an early biomarker of acute exposure to Cd. For exposures lasting more than 4 days, lower doses of Cd (0.1-10 microM) similar to levels encountered in occupational exposure induced a significant increase of the hsp72 level. Because the increase of hsp72 occurs for doses that did not affect cell proliferation, our work supports the idea that its overexpression might be used as a sensitive indicator of occupational exposure to Cd. However, increased resistance to Cd appeared in A549 cells exposed for 1 month and overexpression of hsp72 disappeared simultaneously. It is possible that, in vivo, cell adaptation also occurs throughout chronic exposure to Cd, with a decrease of hsp induction as a consequence. A dose-related increase of MTs was found after 4 days of exposure to Cd concentrations ranging from 0.1 to 10 microM without change of overexpression during chronic exposure, suggesting that MT expression could be a more constant indicator of Cd pollution. Because 0.1 microM Cd (11 microg/L) induces hsp72 expression, showing the presence of damaged proteins, our work suggests that the maximum allowable biologic exposure limit should be lowered.

Pattern of stress protein expression in human lung cell-line A549 after short- or long-term exposure to cadmium.

Exposure to cadmium is associated with the development of pulmonary damage such as emphysema and lung cancer. This metal is also a powerful inducer of stress proteins in many biologic models. The present study was undertaken to evaluate whether an overexpression of the heat shock protein (hsp)72 stress protein, which indicates repair of damaged proteins, could be a sensitive and early biomarker of environmental pollution by Cd. In comparative studies, we examined the effects of exposure to Cd (as CdCl(2)) on the growth rate of the A549 pulmonary cell line, and (by Western blot analyses) on the induction of the hsp72 stress protein and metallothioneins (MTs). CdCl(2) exposure was studied for periods of 2 hr to 1 month. For short-term exposure (2-6 hr) to Cd concentrations higher than 50 microM, an overexpression of hsp72 appeared 6 hr later, suggesting that hsp72 might be considered an early biomarker of acute exposure to Cd. For exposures lasting more than 4 days, lower doses of Cd (0.1-10 microM) similar to levels encountered in occupational exposure induced a significant increase of the hsp72 level. Because the increase of hsp72 occurs for doses that did not affect cell proliferation, our work supports the idea that its overexpression might be used as a sensitive indicator of occupational exposure to Cd. However, increased resistance to Cd appeared in A549 cells exposed for 1 month and overexpression of hsp72 disappeared simultaneously. It is possible that, in vivo, cell adaptation also occurs throughout chronic exposure to Cd, with a decrease of hsp induction as a consequence. A dose-related increase of MTs was found after 4 days of exposure to Cd concentrations ranging from 0.1 to 10 microM without change of overexpression during chronic exposure, suggesting that MT expression could be a more constant indicator of Cd pollution. Because 0.1 microM Cd (11 microg/L) induces hsp72 expression, showing the presence of damaged proteins, our work suggests that the maximum allowable biologic exposure limit should be lowered.

Sensitivity of Escherichia coli (MutT) and human (MTH1) 8-oxo-dGTPases to in vitro inhibition by the carcinogenic metals, nickel(II), copper(II), cobalt(II) and cadmium(II).

The toxicity of Ni(II), Co(II) and Cu(II) in animals, and that of Cd(II) in cultured cells, has been associated with generation of the promutagenic lesion 8-oxo-7,8-dihydroguanine (8-oxoguanine) in DNA, among other effects. One possible source of this base may be 8-oxo-7,8-dihydro-2'-deoxyguanosine-5'-triphosphate (8-oxo-dGTP), a product of oxidative damage to the nucleotide pool, from which it is incorporated into DNA. To promote such incorporation, the metals would have to inhibit specific cellular 8-oxo-dGTPases that eliminate 8-oxo-dGTP from the nucleotide pool. The present study was designed to test such inhibition in vitro on 8-oxo-dGTPases from two different species, the human MTH1 protein and Escherichia coli MutT protein. In the presence of Mg(II), the natural activator of 8-oxo-dGTPases, all four metals were found to inhibit both enzymes. For MTH1, the IC50 values (+/- SE; n = 3-4) were 17 +/- 2 microM for Cu(II), 30 +/- 8 microM for Cd(II), 376 +/- 71 microM for Co(II) and 801 +/- 97 microM for Ni(II). For MutT, they were 60 +/- 6 microM for Cd(II), 102 +/- 8 microM for Cu(II), 1461 +/- 96 microM for Ni(II) and 8788 +/- 1003 microM for Co(II). Thus, Cu(II) and Cd(II) emerged as much stronger inhibitors than Ni(II) and Co(II), and MTH1 appeared to be generally more sensitive to metal inhibition than MutT. Interestingly, in the absence of Mg(II), the activity of the enzymes could be restored by Co(II) to 73% of that with Mg(II) alone for MutT, and 34% for MTH1, the other metals being much less or non-effective. The difference in sensitivity to metal inhibition between the two enzymes may reflect the differences in the amino acid ligands, especially the cysteine ligand, outside their evolutionarily conserved Mg(II)-binding active sites, which might indicate predominantly non-competitive or uncompetitive mechanism of the inhibition. The overall results suggest that inhibition of 8-oxo-dGTPases may be involved in the mechanisms of induction of the 8-oxoguanine lesion in DNA by the metal ions studied, especially the non-redox-active Cd(II) cation.

Changes of actin cytoskeleton during swelling and regulatory volume decrease in cultured astrocytes.

Swelling of cultured astrocytes exposed to hyposmotic medium modified the organization of the filamentous actin (F-actin) cytoskeleton, making the actin network diffuse in the cell body but concentrated at foci corresponding to the tips of the cell projections retracted by swelling. This change was reversible, and, after 2 h, the actin cytoskeleton tended to recover, and cells regained their flat and stellate shape. Cytochalasins B and D (CB and CD, respectively), which disrupt the actin cytoskeleton, did not affect regulatory volume decrease (RVD) or the swelling-activated efflux of Cl- and inositol, although 10 microM CD increased the basal efflux of taurine. The mercurial p-chloromercuribenzenesulfonate (0.5-1 mM), known to disrupt the membrane cytoskeleton in isosmotic conditions, induced a 46, 50, and 38% release of [3H]taurine, 125I, and [3H]inositol, respectively, causing cell shrinkage and retraction of the cytoskeleton. Coincidently, the swelling-stimulated release of [3H]taurine and 125I was reduced by 60 and 30%, respectively. Results of this study do not exclude the possibility that changes in the actin cytoskeleton elicited by swelling are involved in mechanisms of RVD and only indicate that the disruption caused by cytochalasins is unrelated to that process.

Electrophysiological properties of neurones in cultures from postnatal rat dentate gyrus.

Electrophysiological properties of neurofilament-positive neurones in dissociated cell cultures were prepared at postnatal days 4-5 from rat dentate gyrus and studied using the whole-cell patch-clamp technique. These cells expressed a fast-inactivating, 0.5 microM tetrodotoxin-sensitive Na+ current; a high-voltage-activated (HVA) Ca2+ current, which was 30 microM Cd(2+)- and partially 2 microM nicardipine-sensitive; and an inward rectifier current, which was sensitive to extracellularly applied 1 mM Cs+. The outward current pattern was composed of a delayed rectifier-like outward current sensitive to 20 mM tetraethylammonium (TEA) and a fast-inactivating, Ca(2+)-dependent outward current. This transient Ca(2+)-dependent K+ outward current was identified by a subtraction procedure. K+ currents recorded under conditions of blocked Ca2+ currents (after rundown of the HVA Ca2+ current or blocked by extracellularly applied Cd2+) were subtracted from control currents. By comparison with the current pattern of identified dentate granule cells, it is concluded that the investigated cell type originated from interneurones or projection neurones of the dentate hilus.

Characterization of a cadmium-zinc complex in lettuce leaves.

Vegetable food contributes a higher amount of daily cadmium (Cd) intake in humans than food of animal origin. The bioavailability of plant Cd depends on the content of plant zinc (Zn). The mechanism by which increased plant Zn lowers the intestinal absorption of plant Cd could be mediated by changes in the chemical speciation of Cd or Zn in plant edible tissues, including Zn-induced phytochelatin synthesis. To test this hypothesis we investigated the chemical speciation of Cd and Zn in leaf extracts of lettuce grown under 10 microM of Cd accompanied by 0.32 or 31.6 microM Zn in nutrient solution. Gel filtration chromatography of the low- or high-Zn leaf extracts yielded a major low molecular weight Cd-Zn complex that eluted at similar elution volume. Compared to low-Zn leaf extracts, high-Zn leaf extracts contained a higher proportion of Zn incorporated into high molecular weight components, and higher content of the amino acids Cys, Gly, Gly, and Asp in the low molecular weight Cd-Zn complex. The peptides isolated by high performance liquid chromatography (HPLC) of the Cd-Zn complex from the low- or high-Zn leaf extracts did not have an amino acid composition identical to phytochelatins. We concluded that 1. Sequestration of Cd or Zn via phytochelatin does not occur in leaves of lettuce containing levels of those metals representatives of Zn-Cd or Cd-only contaminated crops; and 2. Higher Cys, Glu, Gly, and Asp content in high-Zn than low-Zn leaves could lower Cd absorption in animals fed high-Zn crop diets, by enhancing metallothionein synthesis or changing Cd or Zn speciation in the animal gut.

Mechanism of cadmium-induced cytotoxicity in rat hepatocytes

Exposure of rat hepatocytes to cadmium below 50 microM for a short period (10 min) resulted in cellular acidification. Conversely, exposure to Cd more than 50 microM for a long period (60 min) caused cellular alkalinization accompanied by membrane damage as reflected by decrease in cellular K content and loss of intracellular lactic dehydrogenase. In hepatocytes exposed to 5 microM Cd, a concentration sufficient to induce acidification without cytotoxicity, the metal was preferentially associated with the crude nuclei and cell debris fractions, suggesting an interaction between Cd and cell membranes to cause acidification. Omission of bicarbonate from the incubation medium induced cellular acidification. The presence of Cd in this medium did not potentiate the medium-induced acidification. Mg-ATP (25 microM) induced cellular acidification in relation to an increase in the concentration of cytosolic free Ca. The coexistence of Mg-ATP and Cd at the concentrations which had no effect on cellular pH in the presence of either agants induced cellular acidification. These observations suggest that Cd induced cellular acidification by modulating the process connected with the rise in cytosolic free Ca via interaction with plasma membranes. This acidification had no strong immediate cytotoxic actions but led to subsequent cellular alkalinization accompanied with severe cytotoxicity and membrane breakage.

Depolarization-induced Ca entry via Na-Ca exchange triggers SR release in guinea pig cardiac myocytes.

In mammalian heart muscle, Ca entry through L-type Ca channels is thought to be the primary trigger for the sarcoplasmic reticulum (SR) Ca release, which initiates contraction. The results of this study show that, in guinea pig myocytes with a normal internal Na (10 mM Na in pipette), another trigger mechanisms for SR release and contraction exists. A crucial feature of these experiments was the ability to change rapidly the extracellular environment of a single myocyte so that alterations of intracellular Ca and SR Ca load were minimized for each solution change. We found the following results. 1) A switch to Na-free solution 50 ms before depolarization led to an increase of phasic contraction without increasing L-type Ca current (Ica) or Ca loading of the SR. 2) Although rapid application of 20 microM nifedipine 3 s before a + 10-mV pulse blocked ICa completely, 43 +/- 11 (SE) % of the phasic contraction remained. Similar results were obtained by rapid switching to 150 microM Cd to block ICa. 3) Phasic contraction and ICa had different voltage dependence. With steps to positive potentials there was little ICa but still a substantial phasic contraction. 4) Under action potential conditions, 64.6 +/- 7.9% of the control phasic contraction remained after switching to 20 microM nifedipine to block ICa. 5) The contraction remaining with nifedipine was unaffected by adding 100 microM Ni. Because 100 microM Ni blocks T-type Ca channels, this shows that Ca entry via T-type Ca channels is not involved in triggering SR release. 6) The phasic contraction remaining after a rapid switch to nifedipine was blocked completely by adding 5 mM Ni. Because this concentration of Ni is known to block the Na-Ca exchange, this result suggests that the exchange plays a role in triggering SR release. Taken together, the present results indicate that depolarization-induced Ca entry on the Na-Ca exchange is able to trigger SR release and phasic contraction. This explanation can account for increased phasic contraction after a rapid switch to Na-free solution, persistence of a phasic contraction in the complete absence of ICa, substantial phasic contraction at positive test potentials where there is no ICa, and abolition of nifedipine-resistant contraction by 5 mM Ni.

Recovery potential of hepatocytes from inhibition of albumin secretion by cadmium

The aim of this study was to examine albumin production, a typical liver-specific function, in hepatocytes treated with Cd and to examine the reversibility of the perturbations induced by the toxic metal. Cultures of freshly isolated rat hepatocytes were exposed to increasing amounts of Cd in modified Leibowitz L-15 medium for 20 h; the cells were then allowed to recover by further incubation in Cd-free medium for an additional period of 20 h. The levels of albumin secreted into the extracellular medium were determined by enzyme-linked immunosorbent assay and were found to be reduced by Cd in a concentration-dependent fashion over the first 20 h. Inhibition was seen at Cd concentrations that did not cause any loss of cellular viability (up to 0.5 microM Cd), as judged from the release of lactate dehydrogenase by the cells. After replacement of the exposure medium by Cd-free medium, the same pattern of diminished albumin secretion was obtained, revealing the persistence of the cytotoxic effects when recovery conditions were applied. Moreover, hepatocytes exposed to 0.5 microM Cd for 20 h and processed for visualization of albumin immunoreactive sites using protein A-gold and electron microscopy exhibited very low albumin-specific labeling as compared to the controls (0.6 +/- 0.05 vs. 20.0 +/- 2.6 gold particles/micron2). Intracellular glutathione levels were not significantly changed by Cd either after the initial exposure or after the incubation that followed in control medium. The accumulation of Cd by the cells, as measured by graphite furnace atomic absorption spectrophotometry, was concentration dependent. It remained stable after medium change, indicating that Cd efflux was negligible upon reestablishment of normal conditions. The present data show that the perturbations in albumin metabolism caused by Cd are not readily alleviated after the cells are returned to Cd-free medium, suggesting a limited short-term recovery potential against cytotoxic damage. The data also demonstrate that hepatocyte-specific functions can be used as sensitive indicators for the detection of cellular disturbances by hepatotoxins.

Amplification and rearrangement of a prokaryotic metallothionein locus smt in Synechococcus PCC 6301 selected for tolerance to cadmium.

Metal-tolerant cyanobacteria have been isolated from metal-polluted aquatic environments and also selected in culture, but no genes which confer metal tolerance have been described. To investigate the possibility that amplification of a prokaryotic metallothionein gene (smtA), or rearrangement of the smt locus, could be involved in the development of Cd tolerance in Synechococcus PCC 6301, Cd-tolerant lines were selected by stepwise adaptation of a Synechococcus culture. An increase in smtA gene copy number and the appearance of unique additional smtA restriction fragments (both larger and smaller) were detected in these tolerant lines (tolerant to 0.8 microM Cd, 1.3 microM Cd and 1.7 microM Cd). Stepwise adaptation was repeated by using a culture of Synechococcus PCC 6301 inoculated from a single plated colony to obtain four new lines (tolerant to 1.4 microM Cd, 1.8 microM Cd, 2.6 microM Cd and 3.2 microM Cd). Amplification of the smtA gene and development of unique smtA restriction fragments (larger and smaller) were once again detected in these tolerant lines. Amplification and rearrangement of the smt locus were only detected in the seven Cd-tolerant lines, with no evidence of amplification or rearrangement in the non-tolerant lines from which they were derived. As a control, another gene, psaE, was also monitored in these cell lines. There was no evidence of amplification or rearrangement of psaE in the non-tolerant or any of the Cd-tolerant lines.

Cadmium uptake in Norway spruce (Picea abies (L.) Karst.) seedlings.

Root elongation of Norway spruce (Picea abies (L.) Karst.) seedlings was inhibited in the presence of 5 microM Cd, but was unaffected by a Cd concentration at 0.05 microM. Nutrient solutions labeled with (109)Cd were used to investigate the influence of pH, cations and the metabolic inhibitor 2,4-dinitrophenol (DNP) on the uptake of Cd by roots of intact spruce seedlings. Extracellular Cd was removed by washing the roots, and the relative amounts of Cd in the root apoplast and symplast estimated. In the presence of DNP, Cd uptake was reduced at 0.05 microM (non-toxic) but not at 5 microM Cd (toxic). At 0.05 microM Cd, the uptake of Cd into both the apoplast and symplast was dependent on the pH of the nutrient solution. Lower pH decreased Cd accumulation. Aluminum supplied at 100 or 500 microM lowered the Cd concentrations of both the apoplast and symplast. An increase in Ca or Mg supply reduced the Cd concentration of the apoplast but not of the symplast. In the presence of 5 microM Mn, the concentration of Cd in the symplast decreased by 44% compared to the control (1 microM Mn). High concentrations of Zn or Hg did not affect the Cd concentration of the roots.

The effects of pH on the growth ofChlorella vulgaris and its interactions with cadmium toxicity

The effects of pH alone, and in combination with exposure to 0.89 microM cadmium, on the growth response of the green alga Chlorella vulgaris were evaluated. Acidic (3.0-6.2) and alkaline (8.3-9.0) pH values retarded the growth of this alga. Optimal growth occurred when the pH of the medium was adjusted to values of 7.5 and 8.0. When the cells were exposed to pH adjusted medium plus the presence of 0.89 microM Cd, a value known to reduce population growth by 50% at the control pH of 6.9, the affects were additive at the acidic (3.0-5.0) pH ranges. At alkaline pH values of 8.3-9.0 all toxicity responses could be explained by pH adjustment alone, indicating that additional cadmium toxicity was absent. At pH values of 7.5 and 8.0, cadmium toxicity was mitigated against, and resultant growth at pH 8.0 was at the same enhanced rate as this pH without cadmium.

The toxic action and interactions of copper and cadmium to the marine AlgaDunaliella minuta, in both acute and chronic exposure

The effective concentrations of copper and cadmium which reduced the population growth of Dunaliella minuta by 50% after 96 h of static exposure, were determined to be 7.57 microM Cu and 0.34 microM Cd. Short-term static exposure to both metals indicated that their combined action is antagonistic with respect to growth of this chlorophyte. Additionally, long-term exposure to low levels of Cu or Cd led to the acquisition of tolerance towards Cu and Cd, respectively, and co-tolerance towards Cu.

Effect of Chlordecone (Kepone®) on Calcium Transport Mechanisms in Rat Heart Sarcoplasmic Reticulum*

Previous studies from our laboratory have indicated that chlordecone (Kepone CD), an organochlorine insecticide, inhibited cardiac sodium pump activity and catecholamine uptake suggesting that CD may interfere with cardiac function. Sarcoplasmic reticulum (SR) calcium pump has an important role in myocardial contraction and relaxation, besides Na+ transport. Since CD interferes with cardiac Na+ ion translocases, we have studied CD effects on cardiac SR calcium pump activity. Experiments were carried out both in vitro and in vivo. SR was isolated from heart ventricles of male Sprague-Dawley rats. Cardiac SR Ca2(+)-ATPase. 45Ca-uptake and cAMP as well as calmodulin (CaM) dependent protein phosphorylation were measured. Ca2(+)-ATPase was differentiated into low affinity and high affinity forms by measuring the activity using 50 and 0.7 microM free Ca2(+)-respectively. CD in vitro inhibited 45Ca-uptake by SR in a concentration dependent manner with an IC50 value of 7 microM and SR 45Ca-uptake was totally inhibited at 20-30 microM CD. In agreement with this, both high affinity and low affinity Ca2(+)-ATPases, which are involved in Ca2+ transport across membranes, were also inhibited by CD in a concentration dependent manner with IC50 values of 0.7 and 3.2 microM respectively. Both Ca2(+)-ATPase and 45Ca-uptake by cardiac SR were significantly lower in rats treated with CD (25, 50 or 75 mg/kg) when compared to control rats. cAMP as well as CaM significantly elevated the 32P-binding to SR proteins in vitro to about 70-80%. In the presence of CD, this 32P-binding was reduced, however, not concentration dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

Ca currents in human neuroblastoma IMR32 cells: kinetics, permeability and pharmacology

We have investigated the kinetics, permeability and pharmacological properties of Ca channels in in vitro differentiated IMR32 human neuroblastoma cells. The low-threshold (LVA, T) Ca current activated positive to -50 mV and inactivated fully within 100 ms in a voltage-dependent manner. This current persisted in the presence of 3.2 microM omega-conotoxin (omega-CgTx) or 40 microM Cd and showed a weaker sensitivity to Ni and amiloride than in other neurons. The high-threshold Ca currents (HVA,L and N) turned on positive to -30 mV, and inactivated slowly and incompletely during pulses of 200 ms duration. The amplitude of the HVA currents and the number of 125I-omega-CgTx binding sites increased markedly during cell differentiation. In agreement with recent reports, 6.4 microM omega-CgTx blocked only about 85% of the Ba currents through HVA channels in 50% of the cells. Residual omega-CgTx-resistant currents proved to be more sensitive to dihydropyridines (DHP) than total HVA currents. Bay K 8644 (1 microM) had a clear agonistic action on omega-CgTx-resistant currents and was preferred to other Ca antagonists for identifying HVA DHP-sensitive channels. Compared to the omega-CgTx-sensitive, the DHP-sensitive currents turned on at slightly more negative potentials and showed a weaker sensitivity to voltage. The two HVA currents were otherwise hardly distinguishable in terms of activation/inactivation kinetics, Ca/Ba permeability and sensitivity to holding potentials. This suggests that currently used criteria for identifying multiple types of neuronal Ca channels (T;L,N) may be widely misleading if not supported by pharmacological assays.

A morphometric and x-ray energy dispersive approach to monitoring pH-altered cadmium toxicity inAnabaena flos-aquae

Cadmium toxicity and uptake as influenced by different pH values have been studied in the freshwater cyanobacterium Anabaena flos-aquae, using the techniques of morphometric analysis, x-ray energy dispersive analysis and atomic absorption spectrophotometry. A general reduction in cell dimension, thylakoid surface area, number and volume of polyhedral bodies, polyphosphate bodies, cyanophycin granules, lipid bodies, membrane limited crystalline inclusions, volume and number of wall layers and mesosomes was observed. These reductions were more pronounced in both acidic and alkaline medium than at pH 7.2. At 0.12 microM Cd, the uptake increased with alkaline pH values, and uptake was greater at pH 7.2 than at either acid or alkaline pHs. Lysis of cell wall at 1.18 microM Cd showed the following decreasing trend: pH 4.0 greater than pH 5.5 greater than pH 10.0 greater than pH 9.0 greater than pH 7.2. There was a total loss of lipid bodies at 1.18 microM Cd at all pH values listed. It is suggested that these techniques can be successfully employed for bioassay studies of metal toxicity to algae. In particular, cell wall lysis and loss of lipids by algae are good indicators of pH effects and metal toxicity in the aquatic ecosystem.

Measurement of manganese amelioration of cadmium toxicity inChlorella pyrenoidosa using turbidostat culture

Cadmium (Cd) toxicity and amelioration of Cd toxicity by Mn were measured in Chlorella pyrenoidosa, using turbidostat culture. The responses were measured in terms of the maximum specific growth rate, mu max, of the populations. In turbidostat culture mu max is a dependent variable that can be measured continuously. Cd (as CdCl2.2.5 H2O) was added to control populations at a concentration of 1.8 microM Cd. Toxicity was expressed after a 5 generation lag and resulted in a mu max steady state 62% lower than the initial control after 2 generations. With continued Cd exposure, Mn (as MnCl2.6H2O) was then added stepwise to a concentration of 10.4 microM Mn which caused a rapid, immediate increase in mu max followed by linear increase until a steady-state plateau was reached at a mu max 90% of control. The ameliorative response spanned 20 culture generations. After addition of Mn (10.4 microM), cellular Cd concentration did not change and cellular Mn concentration increased. Increase in mean cell size accompanied Cd exposure and was significantly decreased when supplemented with 10.4 microM Mn. Possible mechanisms of the amelioration are discussed.

Characterization of the effects of ω‐conotoxin GVIA on the responses of voltage‐sensitive calcium channels

1. omega-conotoxin GVIA (omega-CT) caused a potent (IC50 approximately 2nM) but less than maximal (55%) inhibition of [3H]-noradrenaline release from cortical brain slices induced by K+. At 0.1 microM, omega-CT inhibited [3H] gamma-aminobutyric acid (GABA) and [3H]-acetylcholine release by approximately 40%. 2. K+-evoked [3H]-noradrenaline release from cortical brain slices was also characterized with respect to the effects of PN 200-110 (dihydropyridine L-channel antagonist), BAY K8644 (L-channel VSCC agonist), and Cd++ (an inorganic L- and N-channel antagonist). 10 microM Cd++ and 1 microM PN 200-110 inhibited K+-evoked [3H]-noradrenaline release by 52% and 17%, respectively. 10 microM Bay K 8644 enhanced K+-evoked [3H]-noradrenaline release by 22%, and this enhancement was blocked by 1 microM PN 200-110. 3. omega-CT caused a near-maximal inhibition of the electrically evoked twitch responses of the rat vas deferens (IC50 approximately 10 nM) and guinea-pig ileum (IC50 approximately 60 nM), but had no effect on the postjunctional contractile responses of noradrenaline (vas deferens) or carbachol (ileum). At concentrations as high as 1 microM, omega-CT had no effect on the K+-induced contraction of the rat aorta. 4. Neither the equilibrium binding of [3H]-(+)-PN 200-110 nor the allosteric regulation of [3H]-(+)-PN 200-110 binding by tiapamil or diltiazem were altered by omega-CT (0.1 microM). 5. These observations support the notion that the N-type voltage-sensitive calcium channel plays a major role in coupling neuronal excitation with neurotransmitter release.

Subclinical response to cadmium in liver cells

Effects of cadmium (Cd) in vivo and in vitro were studied in the absence of enhanced metallothionein (MT) production and overt Cd toxicity. Such a condition was established by extended oral exposure of male rats to 0.2 mumols Cd/kg and by incubation of isolated hepatocytes with up to 25 microM for 30 min. Subsequently, mitochondrial and extramitochondrial responses to Cd were recorded. Cadmium diminished the activity of cytochrome c oxidase (CYT C OX) by 50% in vivo and by 35% in vitro. In hepatocytes, this was accompanied by increased Cd and decreased protoheme (PrH) in mitochondria. Extramitochondrial PrH and cytochrome P 450 were not significantly altered. In hepatocytes from phenobarbitone pretreated rats, 25 microM Cd decreased CYT C OX but not mitochondrial PrH. Moreover, stimultaneous incubation of hepatocytes with 25 microM Cd and either 2.5 mM dithiothreitol or 5 mM reduced glutathione diminished cellular and mitochondrial Cd and prevented the decrease in CYT C OX but not that in PrH. In contrast, coincubation with either 250 microM L-buthionine-sulfoximine or diethylmaleate, which did not alter Cd uptake, prevented the decrease in PrH but not that in CYT C OX owing to Cd. These results show that Cd exerts mitochondrial alterations in vivo and in vitro in the absence of enhanced MT production. Moreover, Cd effects on CYT C OX and PrH do not seem to be firmly linked.