μM For Cd Research Articles

Zn 2+ and Cd 2+ increase the cyclic GMP level in PC12 cells by inhibition of the cyclic nucleotide phosphodiesterase

In the present study, the influence of the heavy metal ions Cd 2+ and Zn 2+ on cGMP metabolism in the neurosecretory rat pheochromocytoma (PC12) cell line has been investigated. Cadmium and zinc ions showed a concentration-dependent increase of intracellular cGMP levels as determined by radioimmunoassay: a 20-fold increase in cGMP concentration was found after 15 min of incubation with 20 μM Cd 2+, and a 7-fold increase in cGMP was found after incubation with 50 μM Zn 2+ (control: 6.05±2.1 pmol cGMP/mg protein). To obtain further mechanistic informations, the effects of Cd 2+ and Zn 2+ on intracellular 3′,5′-cyclic nucleotide phosphodiesterase have been studied by a high performance liquid chromatography-based phosphodiesterase-assay. The cellular cGMP hydrolysis was found to be inhibited by these ions with an IC 50 value of 6±0.7 μM for Cd 2+ and 13±2.5 μM for Zn 2+. Hence, dose-dependent increase in cellular cGMP content is due to an inhibition of cGMP hydrolysis and not due to an increase in cGMP synthesis. Cd 2+ and Zn 2+ were taken up by PC12 cells as determined by atomic absorption spectroscopy, all measurements were performed in a subtoxic concentration range. Our data illustrate that zinc and cadmium ions are efficient inhibitors of the cGMP-stimulated cyclic nucleotide PDEII in PC12 cells resulting in elevated cellular cGMP concentrations. Therefore, subtoxic doses of these metals may disturb intracellular cGMP/cAMP-signalling pathways leading to an impaired or altered gene expression.

Effects of heavy metals and 3‐methylcholanthrene on expression and induction of CYP1A1 and metallothionein levels in trout (Oncorhynchus mykiss) hepatocyte cultures

AbstractInduction of both CYP1A1 and metallothioneins (MTs) in fish liver is increasingly being used in ecotoxicological studies. The interaction of heavy metals (Cd, Cu, Zn, Pb) with the CYP1A induction response and MT levels was studied in trout (Oncorhynchus mykiss) hepatocyte cultures. Cells were exposed to 3‐methylcholanthrene (3‐MC) or to increasing heavy metal concentrations or to a mixture of both (3‐MC and one heavy metal). Metal cytotoxicity was assessed by the neutral red test. Ranking of toxicity was Cd(II) > Cu(II) > Zn(II) > Pb(II) (EC50: 45, 222, 873, and 945 μM, respectively). CYP1A1 expression was monitored by ethoxyresorufin‐O‐deethylase (EROD) activity as well as by Western and Northern blots. As expected, 3‐MC induced EROD activity in a time‐ and dose‐dependent manner (maximal induction 5 times that of the control at 0.5 μM and after a 72‐h exposure period). These data were confirmed by Western blot (intense band of 55–60 KDa) and Northern blot analyses. Induction caused by 0.5 μM 3‐MC was reduced to less than 50% of control by the concomitant exposure to Cd, Cu, Pb, or Zn (EC50: from 1 μM for Cd(II) to 18 μM for Pb(II)). The MTs were significantly induced in hepatocytes exposed to heavy metals for 24 h. In the presence of 3‐MC (0.5 μM), MT levels were significantly lower than those found in cells treated with metals alone at 24 h only. Our results lead to the conclusion that heavy metals significantly affect CYP expression and that a CYP1A1 inducer (3‐MC) can modulate the induction of MTs. These data have to be taken into consideration in biomarker monitoring.

Expression and Induction of Cyp1A1 in Black Seabream (Spondyliosoma Cantharus) Hepatocyte Cultures: Effects of Heavy Metals

We developed a new method, which allowed black seabream hepatocytes to be maintained in primary-culture for several days. This method was shown to be suitable for studying sea fish CYPs expression and regulation following xenobiotic treatment. After isolation, hepatocytes were directly mixed with a type I collagen gel and culture medium, seeded in 6-well tissue culture plates (2.106 cells/35 mm well) and incubated under atmospheric air at 20°C. After 24 hrs, the cells were exposed for 1, 2 and 3 days to 3-methylcholanthrene (3MC), heavy metals (Cd(II), Cu(II), Pb(II), Zn(II), or 3MC with increasing concentrations of these metals. Cytotoxicity was assessed by the neutral red test: the sequence was Cd(II) > Cu(II) > Pb(II) > Zn (II) (EC50: 57.234, 544 and 722 μM respectively) and appeared to be correlated with the metal solubility. CYP1A1 expression was monitored by EROD activity as well as by Western and Northern blots. 3MC induced the CYP1A1-related EROD activity in a time-and dose-dependent manner. These data were confirmed by Western blot (intense band of 55–60 KDa) and Northern blot analysis. Maximal induction (17-fold over control) was obtained at 1 μM after 72 hrs of exposure. This induction was inhibited by simultaneous exposure to Cd(II), Cu(II), Pb(II) or Zn(II). This effect was dose-dependent and EROD activity was decreased to less than 50% of the control (1 μM 3MC), with EC50 ranging from 1.3 μM for Cd(II) to 16 μM for Zn(II). CYP1A1 protein and mRNA levels were also reduced. CYP1A induction response in fish liver is increasingly being used in ecotoxicological studies. Our results lead to the conclusion that heavy metals significantly affect CYPs expression and therefore have to be taken into consideration in biomarker monitoring.

The SR Ca 2+ ATPase of the Antarctic scallop Adamussium colbecki : cold adaptation and heavy metal effects

Cell calcium is accumulated in intracellular stores by sarco-endoplasmic reticulum Ca2+ ATPases functionally interacting with the membrane lipid environment. Cold adaptations of membrane lipids in Antarctic Sea organisms suggest possible adaptive effects also on sarco-endoplasmic reticulum Ca2+ ATPases. We investigated the SR Ca2+ ATPase of an Antarctic scallop, Adamussium colbecki, by characterising the enzyme activity and studying temperature effects. Ca2+ ATPase, assayed by following ATP hydrolysis, was thapsigargin- and vanadate-sensitive, showed maximum activity under 2 μM Ca2+, 200 mM KCl and pH 7.2, and had a KM for ATP of 22 ± 7 μM. Temperature effects showed an Arrhenius inversion between −1.8 and 0°C, indicating cold adaptation, an Arrhenius break at 10°C, and a collapse above 20°C. A. colbecki accumulates high amounts of cadmium in the digestive gland; heavy metal effects on sarco-endoplasmic reticulum Ca2+ ATPases were therefore tested, finding an IC50 = 0.9 μM for Hg2+ and 3 μM for Cd2+. Finally, SDS-PAGE analysis showed a main band at about 100 kDa, which was identified as sarco-endoplasmic reticulum Ca2+ ATPase after trypsin digestion, and accounted for 60% total protein.

Effects of Cd 2+, Pb 2+ and CH 3Hg + on high voltage-activated calcium currents in pheochromocytoma (PC12) cells: potency, reversibility, interactions with extracellular Ca 2+ and mechanisms of block

Effects of the neurotoxic heavy metals Cd 2+, Pb 2+ and CH 3Hg + on current carried by Ca 2+ ions ( I Ca) through high-voltage activated Ca 2+ channels in nerve growth factor (NGF)-differentiated pheochromocytoma (PC12) cells were examined to characterize possible differences in the mechanism of action of these metals on Ca 2+ channel function. Specifically, the potency and reversibility of effect on I Ca by each metal was examined, as well as the relationship between extracellular [Ca 2+] and potency of block of I Ca by Cd 2+ and Pb 2+. In addition, the effect of each of these metals on Ca 2+ channels when applied to the intracellular side of the membrane was also examined. When extracellular solution contained 20, 10 or 5 mM Ca 2+, the estimated IC 50 values (total metal concentration) for block of I Ca were 15, 10, and 6.5 μM for Cd 2+ and 7.5, 2.0 and 1.1 μM for Pb 2+, respectively. CH 3Hg + (1–10 μM) blocked I Ca (20 mM Ca 2+) in a time- and concentration-dependent manner. When cells were washed with metal-free solutions, block of I Ca by Cd 2+ was reversed rapidly, whereas block by Pb 2+ was reversed only partially, and block of I Ca by CH 3Hg + was not reversed. When Pb 2+ and CH 3Hg + treated cells were washed in metal-free solutions containing 50 μM d-penicillamine (DPEN), block of I Ca by 10 μM Pb 2+ was rapidly and completely reversed, whereas, block of I Ca by 5 μM CH 3Hg + was not reversed. Higher concentrations (500 μM) of 2,3-dimercapto-1-propane sulfonic acid (DMPS) did reverse partially the block of I Ca by 5 and 10 μM CH 3Hg +. When Cd 2+, Pb 2+ or CH 3Hg + was present in the intracellular solution, Ca 2+ channel currents were significantly reduced. These results characterize effects of Cd 2+ on Ca 2+ channels and demonstrate that Cd 2+, Pb 2+ and CH 3Hg + differ in their actions on Ca 2+ channels.

Organic chelation of cadmium using D/‐PDMS: A bioconvecttve test for protective effects

Abstract Cadmium is a major industrial effluent and the cause of itai‐itai disease. As a biological detector for cadmium toxicity, an assay using the motile response of the protozoa species, Tetrahymena pyriformis, has been described. The assay relies on macroscopic bioconvective patterns to score the toxic response, giving a sensitivity better than 1 μM and a toxicity threshold to 5 ( μM for Cd+2. Using pattern formation as the toxicity monitor, the organic chelating agent D/‐PDMS is investigated individually for toxic effects at low dosages (without cadmium) and for poison protection directly (with cadmium). Dosages of 0.1–0.5 μM Di‐PDMS show protective chelation of cadmium and enhance pattern formation capabilities for 1 μM Cd+2.

Bioconvective indicators inTetrahymena:Nickel and copper protection from cadmium poisoning

As a biological detector for cadmium toxicity, an assay using the motile response of the protozoa species, Tetrahymena pyriformis, has been described. The assay relies on macroscopic bioconvective patterns to score the toxic response, giving a sensitivity better than 1 μM and a toxicity threshold to 7 μM for Cd+2 . Using pattern formation as the toxicity monitor, nickel and copper are investigated individually for toxic effects at low dosages without cadmium and for poison protection directly against cadmium. Both 0.01 mM Ni and 0.05 mM Cu show protective competition with cadmium and enhance pattern formation capabilities for 3 μM Cd+2.

Pb 2+ blocks calcium currents of cultured dorsal root ganglion cells

The divalent cation lead (Pb 2+) blocks sustained and transient voltage sensitive calcium channel currents of cultured rat dorsal root ganglion cells. The IC 50 for inhibition of the total peak current evoked by a step depolarization from −80 to 0 mV was 0.6 μM, compared to an IC 50 of 2.2 μM for Cd 2+. The current activated by a depolarization from −40 to 0 mV was inhibited by 50% by 1.0 μM Pb 2+. Low threshold currents activated by a step from −100 to −30 mV were blocked by Pb 2+ at higher concentrations (IC 50=6 μM). The block progressed in the absence of channel activation and showed little voltage dependence. Peak sodium current was reduced by 6.6% at 1 μM Pb 2+ while at 20 μM the peak current was reduced by 40% with marked slowing of the time course of activation. The potassium rectifier current was reduced by 4.1% at 1 μM Pb 2+. Thus, Pb 2+ selectively blocks calcium currents at concentrations in the range of those causing toxicity in man.

Diffusion of calcium, cadmium and mercury in a mucous solution from rainbow trout

1. Diffusion of calcium, cadmium and mercury was studied in a solution of mucus from rainbow trout. Diffusion rates for Cd and Hg were reduced by at least 50% when compared with that in a borate buffer solution. 2. Calcium diffusion rate was unaffected by the presence of mucus. 3. The effect of mucus on the diffusion rates is most likely the consequence of binding of the metals to the mucous proteins. 4. Equilibrium dialysis studies of equimolar concentrations of the metals in a mucous solution demonstrated that about 50% of the Ca, 95% of the Cd and 99% of the Hg remained bound. 5. The dissociation constants ( K d ) for mucus and the metals are: 15 μM for Ca 2+, 0.95 μM for Cd 2+ and 0.33 μM for Hg 2+. 6. The significance of the results for uptake of these metals via the gills is discussed.