Elisabetta Morelli

Characterization of cadmium- and lead-phytochelatin complexes formed in a marine microalga in response to metal exposure.

Phytochelatins (PCn) are thiol-containing peptides with general structure (γ-Glu-Cys)n-Gly enzymatically synthesized by plants and algae in response to metal exposure. They are involved in the cellular detoxification mechanism for their capability to form stable metal-phytochelatin complexes. The speciation of Cd and Pb complexes with phytochelatins has been studied in laboratory cultures of the marine diatom Phaeodactylum tricornutum. An approach based on size-exclusion chromatography (SEC) with off-line detection of phytochelatins, by reverse-phase HPLC, and metal ion, by atomic absorption spectrometry, has been used. The formation of Cd- and Pb-PCn complexes with n-value from 3 to 6 was demonstrated. The metal-PCn complexes formed with Cd appear to be different from those formed with Pb for the number of molecules of peptide involved in the complex and for the amount of the metal ion bound. The chromatographic behaviour of metal-PCn complexes is consistent with Pb-PCn complexes in which only a molecule of peptide binds the metal ion, and with Cd-PCn complexes containing two or more molecules of peptide. The metal/peptide molar ratio in Cd-PCn complexes was higher that in Pb-PCn complexes. The formation of Cd- or Pb-PC2 complexes was not demonstrated, probably for a dissociation during the cellular extract preparation. The effectiveness of phytochelatins in the detoxification of these two metal ions in this alga is discussed.

Properties of phytochelatin-coated CdS nanocrystallites formed in a marine phytoplanktonic alga (Phaeodactylum tricornutum, Bohlin) in response to Cd

Abstract Phytochelatins (PC) are (γ-Glu–Cys)nGly peptides involved in the cellular detoxification mechanism of plants, algae and yeasts for their capability to form stable metal–PC complexes. Phaeodactylum tricornutum exposed to Cd forms Cd–PC complexes in which sulfide ions (S2−) can be incorporated to stabilize PC-coated CdS nanocrystallites. Native CdS particles exhibited differences in their particle size, sulfide content and optical spectroscopic properties. The λmax of the UV transition ranged from 290 to 260 nm. The radii were predicted to vary from 9 to 11 A. The sulfide/Cd molar ratio ranged from 0.89 to 0.26. Low sulfide particles showed propensity to in vitro sulfide mediated accretion. Characterization of these complexes showed that CdS nanoparticles were mainly coated with γ-glutamyl peptides with n value from 3 to 5. The PC2 oligopeptide was not found as coating peptide. CdS particles were stable in the pH range from 8 to 6 and showed half-dissociation at pH 4.9. In vitro reaction with S2− easily converted native, sulfide-free Cd–PC complexes to PC-coated CdS nanocrystallites, but was less effective to restrict particles accretion.

Copper complexing capacity of phytoplanktonic cell exudates

Abstract Copper complexing capacity of cell exudates of Dunaliella salina in natural seawater culture medium was investigated in order to evaluate the influence of this organism on speciation of trace metals in seawater. Seawater samples were collected at 200 m and 2 miles off the coast and immediately filtered. Copper complexing capacity (CC Cu ) and stability constants ( K ′) of related cupric complexes were then measured. They were, respectively, 27.1 × 10 −8 mol l −1 and 0.56 × 10 7 l mol −1 for the samples collected at 200 m and 12.8 × 10 −8 mol l −1 and 6.10 × 10 7 l mol −1 for those collected 2 miles off the coast. A stock culture (20 ml, 10 6 cells ml −1 ) in log-phase was inoculated in 2 l of each sample of filtered natural seawater. The trend of cell influence was estimated on filtered culture medium by measuring CC Cu and K ′ after 1 h, 3 and 7 days. From the results it appears that CC Cu increased with respect to time and this was related to the growth rate, indicating a certain relationship with cell metabolic activity. It can be concluded that a comparison between the culture referring to 200 m and 2 miles, respectively, shows that the former presents a CC Cu two times higher than the latter while the K ′ is ten times higher at 2 miles than that at 200 m.

Speciation of cadmium–γ-glutamyl peptides complexes in cells of the marine microalga Phaeodactylum tricornutum

Phaeodactylum tricornutum responds to Cd exposure by synthesizing phytochelatins (PCn )[ (g-Glu � /Cys)n � /Gly] with n values from 2 to 6. Size exclusion chromatography was coupled with reverse-phase HPLC to follow the formation of complexes between Cd and g-glutamyl peptides in cells exposed to the metal ion during the growth cycle. Five stable complexes (Cd � /PCn n � /2 � /6), in which peptides of unique chain length chelate Cd by formation of thiolate bonds, were identified. The formation of Cd � /PCn complexes in which acid labile sulfide is incorporated (PC-coated CdS crystallites) was also demonstrated. These complexes exhibited luminescence and UV transition at 280 nm. The apparent MW was 8 � /12 kDa and the S 2� /Cd ratio 0.4. The 66% of the total PC g-Glu � /Cys units in these complexes were polymerized as PC4. The incorporation of sulfide into Cd � /PCn complexes enhances the Cd/SCys ratio from 0.6 to 1.6. The kinetics of formation of Cd � /thiols complexes is consistent with an involvement of the glutathione (GSH) in the Cd sequestration mechanism of P. tricornutum through the initial formation of Cd � /GSH complexes. The metal ion is subsequently transferred from GSH to Cd-induced phytochelatins to form more stable Cd � /PCn complexes. # 2002 Elsevier Science Ireland Ltd. All rights reserved.

The biogeochemical cycle of mercury in the Mediterranean

Abstract A study of mercury speciation in waters contributing to the knowledge of its biogeochemical cycle in the Mediterranean is reported. Data are presented on mercury concentrations in particulate and dissolved fractions in waters of the Northern Tyrrhenian Sea. The results show that particulate matter plays a role of primary importance on the’ amount and distribution of this metal in waters.

Polarographic Behavior of Metal Phytochelatin Complexes

The polarographic characteristics of metal-phytochelatin complexes (Me-PC) extracted from the marine alga Phaeodactylum tricornutum were investigated by the use of differential pulse polarography (DPP) at dropping mercury electrode (DME). Four metal-phytochelatin complexes differing for the metal bound, namely Cd-, Pb-, Zn- and Cu-PC, were studied, and their polarographic behavior was compared with that of Cd,Zn-thionein and Zn-thionein of mammalian origin. The polarographic characteristics of Me-PC exhibit a dependence on the metal bound to the peptides and on the pH of the solution. Both the organic and inorganic part of Me-PC are electroactive and give polarographic responses depending on their chemical form in solution. Dissociation and formation processes were investigated. The order of stability of the complexes was K′Cu−PC > K′Pb−PC ≥ K′Cd−PC > K′Zn−PC. The same electrochemical mechanisms were found to characterize both the polarographic behavior of metallothioneins and metal-phytochelatin complexes.

Determination of phytochelatins by cathodic stripping voltammetry in the presence of copper(II)

Abstract The voltammetric behaviour of the copper-phytochelatin (PC) complex in 0.5 M NaCl at pH 8.1 at the hanging mercury drop electrode was investigated. An analytical method that utilizes the current produced, in the presence of Cu(II), by a copper complex with the cysteine groups of the adsorbed PCs, was developed. At a Cu(II) concentration of 2.5 × 10 −6 M, sensitive measurements can be achieved by using differential pulse cathodic stripping voltammetry. The effects of the experimental parameters such as preconcentration time, adsorption potential and copper concentration on the peak current are discussed. The stripping current was found to be proportional to the total concentration of thiol groups of phytochelatins (SH PC ). The sensitivity was 8 × 10 −3 nA s −1 nM −1 . Using an accumulation time of 300 s at −0.4 V, an SH PC concentration of 1 nM can be detected.

Susceptibility of insulinoma cells to cadmium and modulation by L-type calcium channels.

Cadmium (Cd), a toxic metal that induces apoptosis and necrosis in a variety of cells, accumulates in pancreas and may be a cause of diabetes in humans. In the insulinoma cells line HIT-T15 (HIT), we measured internal calcium (Ca) and Cd levels by the fluorescent dye Fura-2 and confirm that L-type voltage-dependent calcium channels (VDCC) play a major role in glucose response and represent a pathway of Cd influx in these cells. Therefore we examined the role of VDCC in acute Cd poisoning by comparing its accumulation and cytotoxic effect in HIT cells and in epithelial-like VDCC-free HeLa cells. Cultures were incubated with 10–300 μM Cd for 15 min–6 h. While negligible at the end of the treatment, HIT cell death was evident after 18–24 h, and it was time-, dose- and serum-dependent. Short (≤60 min) Cd treatments with lower doses (≤100 μM in serum-free medium) induced delayed apoptotic cell death, as demonstrated by DNA fragmentation on agarose gels and segmentation of DAPI-stained nuclei. Longer incubations and/or higher concentrations caused mainly necrosis. The same treatments were largely harmless in HeLa cells, in which neither death nor DNA fragmentation was observed. The Ca antagonist nimodipine was capable to prevent HIT cell death at lower doses of Cd and to restore the apoptotic condition at higher doses, indicating that reduction of Cd flux through VDCC modulates Cd toxicity. These data demonstrate a specific sensitivity to Cd of insulinoma cells that can be significant for pancreatic β-cell pathology.

Cadmium induced phytochelatins in marine alga Phaeodactylum tricornutum: effect of metal speciation

ABSTRACTThe production of phytochelatins (PCs) in the marine diatom Phaeodactylum tricornutum in response to cadmium has been studied as a function of the exposure time, concentration and chemical speciation. The results agree with the role of PCs in the detoxification and homeostasis of Cd in this alga. The chemical speciation of the metal was found to influence the synthesis of PCs. The use of Cd ion buffer solutions shows that the induction of PCs is controlled by the free ion rather than by the total or chelated Cd in solution.

Adsorptive stripping voltammetry of lumichrome in sea water at the static mercury drop electrode

Abstract The adsorptive voltammetric behaviour of lumichrome (7,8-dimethylalloxazine), the main product of riboflavin photolysis, was investigated in sea water. The effect of experimental parameters such as preconcentration time and potential, pH of solution and mass transport conditions on the stripping response are discussed. Sensitive measurements can be achieved after controlled adsorption followed by square wave voltammetry. The stripping peak current was found to be proportional to the bulk concentration of lumichrome. The sensitivity was 5 × 10 −2 nA nM −1 s −1 , the reproducibility was 6% at a concentration of 3.6 × 10 −9 M lumichrome and the detection limit was 100 pM. Using an accumulation time of 600 s at −0.4 V, this technique allows the direct determination of lumichrome naturally present in raw sea water samples. The value found was about 200 pM. The concentration of lumichrome was also determined in the same samples using liquid chromatography after a solid-phase preconcentration step. Good correlations between voltammetric and chromatographic results were found.