Application of 2,4,5-tris(2-pyridyl)imidazole as ‘turn-off’ fluorescence sensor for Cu(II) and Hg(II) ions and in vitro cell imaging

The 2,4,5-tris(2-pyridyl)imidazole ( L ) molecule has been evaluated as a probe for dual sensing of Hg²⁺ and Cu²⁺ ions in EtOH/HEPES buffer medium (5 mM, pH = 7.34, 1:1, v/v). Probe L shows a good sensitive and selective turn-off response in the presence of both Hg²⁺ and Cu²⁺ ions, which is comprehensible under long UV light. The probe can detect Cu²⁺ ion in the pH range 3–11 and Hg²⁺ ion in pH 6–8. The limit of detection for Cu²⁺ (0.77 μM) is well under the allowable limit prescribed by the United States Environmental Protection Agency. Two metal (Cu²⁺/Hg²⁺) ions are needed per L for complete fluorescence quenching. The probe shows marked reversibility on treatment with Na₂EDTA, making the protocol more economical for practical purposes. Paper strip coated with the L solution of EtOH can detect the presence of Cu²⁺ and Hg²⁺ ions in the sample using visible quenching of the fluorescence intensity. Density functional theory–time-dependent density functional theory (DFT–TDDFT) calculations support experimental observations, and d-orbitals of Cu²⁺/Hg²⁺ provide a nonradiative decay pathway. Cell imaging study using HDF and MDA-MB-231 cells also supported the viability of L in detecting Cu²⁺ and Hg²⁺ ions in living cells.     

Binding of mercuric and other heavy metal ions by microbial growth media

Ion-specific electrodes were used to study the binding of Hg²⁺, Pb²⁺, Cu²⁺, and Cd²⁺ ions to widely used bacterial growth media (Nutrient broth, trypticase soy broth, the medium of Foot and Taylor [6] and of Nelsonet al.[12]) and to media components [yeast extract, peptone, tryptone, proteose peptone, and casamino acids (acid hydrolyzed casein)]. Volatilization of Hg²⁺ from aqueous solutions could be prevented by any of the growth media or their components. All media bound large amounts of Hg²⁺, Pb²⁺, and Cu²⁺, but much less Cd²⁺. Of the media components, casamino acids showed the most binding activity for all metal ions; the relative affinity of other media components to different ions varied with the cation studied. In general, the Irving-Williams [8] series for cation affinity to organic ligands was followed: Hg²⁺>Pa²⁺ Cu²⁺ Cd²⁺.After adding 20 ppm of Hg²⁺, Pb²⁺, or Cu²⁺ (concentrations inhibitory to the growth of most microorganisms) to the growth media, 80 ppb or less remained as free cations in the solution. This might suggest that such ions enter bacterial cells as organic complexes, or that bacterial cells can compete successfully with growth media for the bound ions.     

Structure of Cotylenol,the Aglycone of the Cotylenins Leaf Growth Substances

A potassium chromate-tolerant bacterium was isolated from activated sludge, and the bacterium was identified as Pseudomonas ambigua G–1. The bacterium tolerated up to 2000 ppm of Cr⁶⁺, 1700 ppm of Cu²⁺ and 200 ppm of Cd²⁺, but did not tolerate Hg²⁺. Chemical analysis indicated 86.5% uptake in the soluble fraction and 13.5% uptake in the insoluble fraction of cells. Chromate uptake distribution in the soluble fraction indicated 28.9% in microsomal fraction and 78.1% in supernatant fraction, Chromate distribution in the insoluble fraction showed 61% in lipid-fraction and 21% in polyphosphate-polysaccharidefraction. Chromate inhibited the syntheses of protein, DNA in soluble fraction and RNA in microsomal fraction.     

Copper and mercury induced oxidative stresses and antioxidant responses of Spirodela polyrhiza (L.) Schleid

Duckweed is recognized as a phytoremediation aquatic plant due to the production of large biomass and a high level of tolerance in stressed conditions. A laboratory experiment was conducted to investigate antioxidant response and mechanism of copper and mercury tolerance of S. polyrhiza (L.) Schleid. To understand the changes in chlorophyll content, MDA, proline, and activities of ROS-scavenging enzymes (SOD, CAT, GPOD) during the accumulation of Cu⁺² and Hg⁺², S. polyrhiza were exposed to various concentrations of Cu⁺² (0.0–40 μM) and Hg⁺² (0.0–0.4 μM). antioxidant activity initially indicated enhancing trend with application of 10 μM Cu⁺²; 0.2 μM Hg⁺² (SOD), of 20 μM Cu⁺²; 0.2 μM Hg⁺² (CAT) and of 10 μM Cu⁺²;0.2 μM Hg⁺² (GPOD) and then decreased consistently up to 40 μM Cu⁺² and 0.4 μM Hg⁺². In the experiment chlorophyll and frond multiplication initially showed increasing tendency and decreased gradually with the application of increased metal concentration. Application of heavy metal has constantly enhanced proline and MDA content while the maximum increase was observed with the application of 40 μM Cu; 0.4 μM Hg for proline and MDA respectively. The upregulation of antioxidant enzymes and proline reveals that S. polyrhiza has strong biochemical strategies to deal with the heavy metal toxicity induced by the accumulation of Cu⁺² and Hg⁺².     

Multifunctional peptide‐based fluorescent chemosensor for detection of Hg2+, Cu2+ and S2− ions

A novel multifunctional fluorescent peptide sensor based on pentapeptide dansyl‐Gly‐His‐Gly‐Gly‐Trp‐COOH (D‐P5) was designed and synthesized efficiently using Fmoc solid‐phase peptide synthesis (SPPS). This fluorescent peptide sensor shows selective and sensitive responses to Hg²⁺ and Cu²⁺ among 17 metal ions and six anions studied in N‐2‐hydroxyethylpiperazine‐N‐2‐ethane sulfonic acid (HEPES) buffer solution. The peptide probe differentiates Hg²⁺ and Cu²⁺ ions by a ‘turn‐on’ response to Hg²⁺ and a ‘turn‐off’ response to Cu²⁺. Upon addition of Hg²⁺ or Cu²⁺ ions, the sensor displayed an apparent color change that was visible under an ultraviolet lamp to the naked eye. The limits of detection (LOD) of DP‐5 were 25.0 nM for Hg²⁺ and 85.0 nM for Cu²⁺; the detection limits for Cu²⁺ were much lower than the drinking water maximum contaminant levels set out by the United States Environmental Protection Agency (USEPA). It is noteworthy that both D‐P5‐Hg and D‐P5‐Cu systems were also used to detect S^2? successfully based on the formation of ternary complexes. The LODs of D‐P5‐Hg and D‐P5‐Cu systems for S^2? were 217.0 nM and 380.0 nM, respectively. Furthermore, the binding stoichiometry, binding affinity and pH sensitivity of the probe for Hg²⁺ and Cu²⁺ were investigated. This study gives new possibilities for using a short fluorescent peptide sensor for multifunctional detection, especially for anions.     

Fixation of CO2 and incorporation of thymidine under heavy metal stress inClosterium moniliferum

The unicellular green algaClosterium moniliferum was sensitive to the action of divalent Ni, Cu, Hg and Cd chlorides both in CO₂ fixation and in thymidine incorporation into DNA. At 0.08 ppm, Cd²⁺ was the most potent inhibitor (86 % inhibition of both processes), followed by Hg²⁺ and Cu²⁺, and finally by Ni²⁺, thymidine incorporation being generally more affected than CO₂ fixation.     

Naked eye sensing of toxic metal ions in aqueous medium using thiophene‐based ligands and its application in living cells

Thiophene‐based diimine (R1) and monoimine (R2) were synthesized in a single step, and their cation binding affinity was tested using colorimetric and UV–vis spectral studies. R1 selectively shows a colorimetric turn‐on response for Pb²⁺, Hg²⁺ ions and colorimetric turn‐off with Sn²⁺ ions, and R2 shows visual response for Cu²⁺ and Hg²⁺ over other examined metal ions in aqueous medium. R1 forms 1:1 complex with Pb²⁺, Hg²⁺, and Sn²⁺ and exhibits fluorescence quenching, whereas R2 shows 2:1 complex with Hg²⁺, Cu²⁺ and shows fluorescence enhancement. The structural and electronic properties of the sensors and their metal complexes were also investigated using Density Functional Theory calculations. R2 was also successfully demonstrated as a fluorescent probe for detecting Cu²⁺ ions in living cells. Copyright © 2014 John Wiley & Sons, Ltd.     

Heavy metal interference with growth hormone signalling in trout hepatoma cells RTH-149

We have studied the effects of heavy metals (Hg²⁺, Cu²⁺, Cd²⁺) on growth hormone (GH) activation of tyrosine kinase and Ca²⁺ signaling in the trout (Oncorhynchus mykiss) hepatoma cell line RTH-149. Molecular cloning techniques using primer designed on Oncorhynchus spp. growth hormone receptor (GHR) genes allowed to isolate a highly homologous cDNA fragment from RTH-149 mRNA. Thereafter, cells were analysed by Western blotting or, alternatively, with Ca²⁺ imaging using fura-2/AM. Exposure of cells to ovine GH alone produced a stimulation of the JAK2/STAT5 pathway and intracellular free Ca²⁺ variations similar to what has been observed in mammalian models. Cell pre-exposure to Cu²⁺, Hg²⁺ or Cd²⁺ affected cell response to GH by enhancing (Cu²⁺) or inhibiting (Cd²⁺) the phosphorylation of JAK2 and STAT5. Heavy metals induced the activation of the MAP kinase p38, and pre-exposure to Hg²⁺ or Cu²⁺ followed by GH enhanced the effect of metal alone. Image analysis of fura2-loaded cells indicated that pre-treatment with Hg²⁺ prior to GH produced a considerable increase of the [Ca²⁺]_i variation produced by either element, while using Cu²⁺ or Cd²⁺ the result was similar but much weaker. Data suggest that heavy metals interfere with GH as follows: Hg²⁺ is nearly ineffective on JAK/STAT and strongly synergistic on Ca²⁺ signaling; Cu²⁺ is activatory on JAK/STAT and slightly activatory on Ca²⁺; Cd²⁺ is strongly inhibitory on JAK/STAT and slightly activatory on Ca²⁺; heavy metals could partially activate STAT via p38 independently from GH interaction.Published online: March 2005     

A sugar-aza-crown ether-based fluorescent sensor for Cu and Hg ions

A sugar-aza-crown ether (SAC)-based fluorescent sensor 4 was prepared. It contains a pyrene as the fluorophore and its fluoroionophoric properties toward transition metal ions were investigated. Chemosensor 4 exhibits highly selective recognition toward Cu²⁺ and Hg²⁺ ions among a series of tested metal ions in methanol solution. The association constants for 4*Cu²⁺ and 4*Hg²⁺ in methanol solution were calculated to be 7.4 × 10¹ M⁻¹ and 4.4 × 10³ M⁻¹, respectively. Chemosensor 4 formed complexes with the Cu²⁺ or Hg²⁺ ion at a 1:1 ligand-to-metal ratio with a detection limit of 1.3 × 10⁻⁴ M Cu²⁺ and 1.26 × 10⁻⁵ M Hg²⁺, respectively.     

Multimode selective detection of mercury by chiroptical fluorescent sensors based on methionine/cysteine

Two multimode Hg(II) sensors, L‐MethBQA and L‐CysBQA, were obtained by fusing methionine or S‐methyl cysteine, into a bis‐quinolyl amine‐based chiral podand scaffold. Quinolyl groups serve as the fluorophore and possess nitrogen lone pairs capable of chelating metal ions. On exposure to Hg²⁺ or Zn²⁺, these sensors show signal enhancement in fluorescence. However, Cu²⁺ quenches their fluorescence in 30:70 acetontrile/water. L‐CysBQA complexes with Hg²⁺, producing an exciton‐coupled circular dichroism spectrum with the opposite sign to the one that is produced by Cu²⁺ or Zn²⁺ complexation. L‐CysBQA binds Hg²⁺ more strongly than Zn²⁺ and is shown to differentiate Hg²⁺ from other metal ions, such as Zn²⁺, Cu²⁺, Ni²⁺, and Pb²⁺, exceptionally well. The synergistic use of relatively soft sulfur, quinoline‐based chiral ligands and chiroptically enhanced fluorescence detection results in high sensitivity and selectivity for Hg²⁺. Chirality, 2011. © 2011 Wiley‐Liss, Inc.     

Hg2+-induced leakage of electrolytes and inhibition of NO3 − utilization inNostoc calcicola

Summary The effect of mercury (Hg²⁺) in the absence and presence of methylmercury (CH₃Hg⁺), cadmium (Cd²⁺), copper (Cu²⁺), nickel (Ni²⁺) and calcium (Ca²⁺) on Nostoc calcicola Bréb. has been studied in terms of electrolyte leakage, NO₃ ^– uptake and in vivo nitrate reductase (NR) activity to discover any possible correlation among such parameters under Hg²⁺ stress. Leakage of electrolytes from Hg²⁺-treated cyanobacterial cells was directly proportional to Hg²⁺ concentrations and exposure time. In comparison to NO₃ ^– uptake, an about 60-fold slower rate of NR activity was observed in the untreated cultures, the former being five times more Hg²⁺-sensitive. A non-competitive synergistic interaction of Hg²⁺ with CH₃Hg⁺ or Cd²⁺ and antagonistic with that of Ni²⁺ or Ca²⁺ has been observed for both the processes of NO₃ ^– utilization. The antagonistic interaction of Cu²⁺ with Hg²⁺ in terms of NO₃ ^– uptake and synergistic with respect to NR activity, has been attributed to the dual bonding preference of Cu²⁺ for cellular ligands. These findings suggest that (a) a statistically significant correlation exists among such parameters; (b) Hg²⁺ predominantly attacks the cyanobacterial cell membrane; (c) Hg²⁺ inhibits NO₃ ^– utilization; (d) the presence of other cations increases or decreases the inhibitory actions of Hg²⁺.     

Improvement of ganoderic acid and Ganoderma polysaccharide biosynthesis by Ganoderma lucidum fermentation under the inducement of Cu2+

The cell growth and total accumulation of bioactive metabolites were significantly improved by Cu²⁺ addition during the submerged fermentation of medicinal mushroom Ganoderma lucidum. A mathematical model, constructed by response surface methodology combination with full factorial design, was applied to study the synergic effect of Cu²⁺ addition concentration and addition time. The optimal Cu²⁺ inducement strategy for the cell growth were different from those for the biosynthesis of ganoderic acid (GA) and Ganoderma polysaccharide. A multiple additions strategy of Cu²⁺ by adding each 1 mM Cu²⁺ on day 2, 6, 8 and 2 mM Cu²⁺ on day 4 was developed to enhance total accumulation of GA and extracellular polysaccharides. The highest GA content reached 3.0 ± 0.1 mg per 100 mg DW, which was increased by 76.5% and 33.9% compared with the control without Cu²⁺ addition and the peak value predicted by the constructed mathematical model, respectively. While, relatively higher addition concentration of Cu²⁺ (i.e., 5 mM) on the culture of day 4 led to higher content and total production of intracellular polysaccharides. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Probing the coordination properties of glutathione with transition metal ions (Cr2+,Mn2+,Fe2+,Co2+, Ni2+,Cu2+,Zn2+,Cd2+,Hg2+) by density functional theory

Complexes formed by reduced glutathione (GSH) with metal cations (Cr²⁺, Mn²⁺,Fe²⁺,Co²⁺,Ni²⁺,Cu²⁺,Zn²⁺,Cd²⁺,Hg²⁺) were systematically investigated by the density functional theory (DFT). The results showed that the interactions of the metal cations with GSH resulted in nine different stable complexes and many factors had an effect on the binding energy. Generally, for the same period of metal ions, the binding energies ranked in the order of Cu²⁺>Ni²⁺>Co²⁺>Fe²⁺>Cr²⁺>Zn²⁺>Mn²⁺; and for the same group of metal ions, the general trend of binding energies was Zn²⁺>Hg²⁺>Cd²⁺. Moreover, the amounts of charge transferred from S or N to transition metal cations are greater than that of O atoms. For Fe²⁺,Co²⁺,Ni²⁺,Cu²⁺,Zn²⁺,Cd²⁺ and Hg²⁺ complexes, the values of the Wiberg bond indices (WBIs) of M-S (M denotes metal cations) were larger than that of M-N and M-O; for Cr²⁺ complexes, most of the WBIs of M-O in complexes were higher than that of M-S and M-N. Furthermore, the changes in the electron configuration of the metal cations before and after chelate reaction revealed that Cu²⁺, Ni²⁺,Co²⁺ and Hg²⁺ had obvious tendencies to be reduced to Cu⁺,Ni⁺,Co⁺ and Hg⁺ during the coordination process.     

Synthesis, characterization, fluorescence and computational studies of new Cu, Ni and Hg complexes with emissive thienylbenzoxazolyl-alanine ligands

The interaction of four fluorescent compounds containing thiophene and benzoxazole moieties combined with an alanine residue with alkaline, alkaline-earth, transition and post-transition metal ions was explored. The highly fluorescent heterocyclic alanine derivatives are strongly quenched in the solid state after complexation with the paramagnetic metal ions Cu²⁺ and Ni²⁺, and with the diamagnetic Hg²⁺. Absorption and steady-state fluorescence titrations reveal a selective interaction with Cu²⁺, Ni²⁺ and Hg²⁺. In all cases the formation of mononuclear or dinuclear metal complexes in solid state and in solution are postulated. DFT calculations on the mercury(II) complexes confirm the formation of dinuclear species. Our results suggest that one metal ion is coordinated by the chelate group formed by the amine and the protonated carboxylic groups present in the amino acid residue while a second metal ion is directly linked to the chromophore. As parent compound, L4 shows no interaction with Cu²⁺ and Ni²⁺ salts. However, the interaction with Hg²⁺ induces a strong quenching and a red shift of the fluorescence emission.     

Copper blocks V‐ATPase activity and SNARE complex formation to inhibit yeast vacuole fusion

The accumulation of copper in organisms can lead to altered functions of various pathways and become cytotoxic through the generation of reactive oxygen species. In yeast, cytotoxic metals such as Hg⁺, Cd²⁺ and Cu²⁺ are transported into the lumen of the vacuole through various pumps. Copper ions are initially transported into the cell by the copper transporter Ctr1 at the plasma membrane and sequestered by chaperones and other factors to prevent cellular damage by free cations. Excess copper ions can subsequently be transported into the vacuole lumen by an unknown mechanism. Transport across membranes requires the reduction of Cu²⁺ to Cu⁺. Labile copper ions can interact with membranes to alter fluidity, lateral phase separation and fusion. Here we found that CuCl₂ potently inhibited vacuole fusion by blocking SNARE pairing. This was accompanied by the inhibition of V‐ATPase H⁺ pumping. Deletion of the vacuolar reductase Fre6 had no effect on the inhibition of fusion by copper. This suggests that Cu²⁺ is responsible for the inhibition of vacuole fusion and V‐ATPase function. This notion is supported by the differential effects of chelators. The Cu²⁺‐specific chelator triethylenetetramine rescued fusion, whereas the Cu⁺‐specific chelator bathocuproine disulfonate had no effect on the inhibited fusion.     

Copper and Manganese Increase Resistance of Clusterbean to Root Rot Caused by Rhizoctonia

Clusterbean seedlings growing in soil inoculated with Rhizoctonia were treated with either 5 or 10 ppm Cu²⁺ and Mn²⁺. Soil was inoculated by pretreatment with 250 mg (wet weight) of Rhizoctonia inoculum. A similar set of plants was maintained in uninoculated soil. Root rot incidence of plants treated with Cu²⁺ 5 ppm, Cu²⁺ 10 ppm, Mn²⁺ 5 ppm and Mn²⁺ 10 ppm was 26.6, 30.5, 11.8 and 29.2% less than the inoculated control, respectively. Inoculation with Rhizoctonia reduced chlorophyll, non‐structural carbohydrate and in vitro dry matter digestibility (IVDMD) content compared with uninoculated ones. Oxidative enzymes activities (polyphenol oxidase, peroxidase, phenylalanine ammonia lyase and tyrosine ammonia lyase), crude protein, phenolic content, structural components (acid detergent fibre, cellulose and lignin), silica, macronutrients and micronutrients increased in inoculated seedlings and this increase was further heightened by the Cu²⁺ 10 ppm treatment compared with the Cu²⁺ 5 ppm, Mn²⁺ 5 ppm and Mn²⁺ 10 ppm treatments in response to fungal invasion. It was concluded that the Cu²⁺ 10 ppm treatment may be an effective soil nutrient to provide enhanced resistance of clusterbean plants to root rot (fungal) diseases.     

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.     

Effect of neurotoxic divalent cations on the activity of the intrinsic nerve ending membrane-associated sialidase of bovine brain

Exposure to Hg²⁺ below 10 M destroys synaptosomal membrane-associated sialidase of bovine brain in situ. Inhibition by Cu²⁺ occurs only at relatively higher concentrations, and is demonstrable after the synaptosomal nembrane preparation has been presaturated with Cu²⁺. Pb²⁺ does not inhibit enzymatic activity. Hg²⁺ does not exert a significant effect on the free energy of association of monomeric brain gangliosides into aggregates, or on the stability of the aggregate forms, as estimated by ultracentrifugal analysis of the ion-independent moment of ganglioside micelles as a function of concentration. Hg²⁺ inhibits synaptic membrane sialidase acting both in situ on the native sialocompounds in the membrane, or on exogenous ganglioside. Kinetic analyses of the exogenous activity in membranes exposed to Hg²⁺ reveal loweredV _max values but no substantial change inK _m for synaptosomal membrane gangliosides. These findings suggest that the powerful inhibitory effect exerted by Hg²⁺ on nerve ending membrane sialidase is enzyme directed, not substrate directed. It may be postulated that part of the neurotoxic effect of low levels of Hg²⁺ stems from an interference with synaptic metabolism by the destruction of membrane-associated sialidase. This enzyme can serve the purpose of modulation of synaptic negative charge density by releasing bound, strongly anionic, sialic acid from highly concentrated sialocompounds in the membrane.Author to whom correspondence should be sent.     

The behaviors of metal ions in the CdTe quantum dots–H2O2 chemiluminescence reaction and its sensing application

The behaviors of 15 kinds of metal ions in the thiol‐capped CdTe quantum dots (QDs)–H₂O₂ chemiluminescence (CL) reaction were investigated in detail. The results showed that Ag⁺, Cu²⁺ and Hg²⁺ could inhibit CdTe QDs and H₂O₂ CL reaction. A novel CL method for the selective determination of Ag⁺, Cu²⁺ and Hg²⁺ was developed, based on their inhibition of the reaction of CdTe QDs and H₂O₂. Under the optimal conditions, good linear relationships were realized between the CL intensity and the logarithm of concentrations of Ag⁺, Cu²⁺ and Hg²⁺. The linear ranges were from 2.0 × 10^?6 to 5.0 × 10^?8 mol L^?1 for Ag⁺, from 5.0 × 10^?6 to 7.0 × 10^?8 mol L^?1 for Cu²⁺ and from 2.0 × 10^?5 to 1.0 × 10^?7 mol L^?1 for Hg²⁺, respectively. The limits of detection (S/N = 3) were 3.0 × 10^?8, 4.0 × 10^?8 and 6.7 × 10^?8 mol L^?1 for Ag⁺, Cu²⁺ and Hg²⁺, respectively. A possible mechanism for the inhibition of CdTe QDs and H₂O₂ CL reaction was also discussed. Copyright © 2009 John Wiley & Sons, Ltd.     

Comparison of Physiological Changes in Euglena gracilis During Exposure to Heavy Metals of Heterotrophic and Autotrophic Cells

The effect of different concentrations of Hg²⁺, Cd²⁺, and Pb²⁺ on ultrastructure, growth, respiration, photosynthesis, chlorophyll content, and metal accumulation in Euglena gracilis was examined. The toxicity of the heavy metals was dependent on the culture medium used and whether cells were grown in the dark or under illumination. Hg²⁺ was the most toxic metal, which showed effects at a concentration as low as 1.5 μM; Cd²⁺ showed an intermediate toxicity (effects observed above 50 μM); and Pb²⁺ was almost ineffective up to 1 mM. Cells grown for several weeks in the dark, in the presence of 1.5 μM Hg²⁺ showed a reduced sensitivity to subsequent exposure to Cd²⁺ or Pb²⁺. The Hg²⁺-pretreated cells also presented an enhanced capacity to accumulate other metals. In comparison, light-grown cells showed a greater Cd²⁺ accumulation, but a lower Pb²⁺ uptake than Hg²⁺-pretreated dark-grown cells. Pretreatment of light-grown cells with Hg²⁺ did not enhance the accumulation of Cd²⁺. These results suggest that the capacity to tolerate heavy metals by Euglena may have mechanistic differences when cells are grown in the dark or under illumination.