Cadmium accumulation in the chloroplast of Euglena gracilis

Intracellular distribution of Cd, cysteine, glutathione, and Cd-induced thiol peptides in Euglena gracilis cultured under photoheterotrophic conditions was studied. After 3 days of culture with 0.2 m M CdCl₂, 62% of the Cd accumulated by cells was equally distributed between the cytosolic and chloroplastic fractions. However, after 8 days, metal content increased in the crude chloroplastic fraction to 40% of total and decreased to 19% in the cytosol; in Percoll-purified chloroplasts the estimated content of Cd raised to 62%. Accumulation of Cd in chloroplasts could be mediated by a transporter of free Cd²⁺, since uptake of added CdCl₂ in isolated chloroplasts exhibited a hyperbolic type of kinetics with a K_m of 57 µ M and V_max of 3.7 nmol (mg protein)⁻¹ min⁻¹. The contents of cysteine and glutathione markedly increased in both chloroplasts (7–19 times) and cytosol (4–9 times) by exposure to Cd²⁺, although they were always higher in the cytosol. Thiol-containing peptides induced by Cd were mainly located in the cytosol after 3 days, and in the chloroplasts after 8 days of culture. The data suggested that Cd was compartmentalized into chloroplasts in a process that may involve the transport of free Cd and the participation of thiol-peptides.     

Simian virus 40-transformed metallothionein null cells showed increased sensitivity to cadmium but not to zinc, copper, mercury or nickel

Primary cultured embryonic cells derived from mice with disrupted metallothionein (MT) I and II genes and from control mice were transformed with a plasmid encoding the simian virus 40 (SV40) large T antigen. The resulting MT-/- and MT+/+ cell strains showed similar cell morphology, cell cycle and no significant differences in glutathione levels or in the activities of glutathione-related enzymes and antioxidant enzymes. The MT-/- cells were more sensitive to Cd than MT+/+ cells, though no increase in the sensitivity to Zn, Cu, Hg or Ni were observed in MT-/- cells. MT+/+ cells accumulated more Cd than MT-/- cells but showed less lesion, suggesting the role of MT induced by Cd in MT+/+ cells as a scavenger of toxic Cd ion. These results suggest a dominant protective role of MT against Cd compared with other metals. SV40-transformed MT-/- cells seem to be a useful tool for the investigation of cellular function of MT.     

Cadmium-dependent generation of reactive oxygen species and mitochondrial DNA breaks in photosynthetic and non-photosynthetic strains of Euglena gracilis

The photosynthetic strain Z of Euglena gracilis is more susceptible to cadmium chloride (Cd) than the non-photosynthetic strain SMZ. We investigated the correlation of intracellular reactive oxygen species (ROS) levels with Cd-induced cellular damage. Flow cytometry with dihydrorhodamine 123 showed that strain Z generated higher levels of ROS, probably H(2)O(2) and/or ONOO(-), than strain SMZ, and that this difference between the two strains became more pronounced with increasing Cd dose. The levels of ROS increased at cytotoxic concentrations of Cd, at over 10 microM Cd for Z and 50 microM Cd for SMZ. These results show an association of Cd cytotoxicity with ROS generation. Considering that strain SMZ is non-photosynthetic, the higher levels of ROS in strain Z might be due to blockage of photosynthetic electron flow by Cd. Using terminal deoxyribonucleotidyl transferase-mediated dUTP nick end-labeling analysis in combination with 4',6-diamidino-2-phenylindole, dihydrochloride staining, we observed DNA breaks in the mitochondria of both strains after Cd exposure. The results suggest that the mitochondrion is the primary target organelle of Cd in E. gracilis cells.     

In Euglena gracilis, a heat-shock protein related to hsc73 is constitutive and stress inducible

Using monoclonal antibodies directed against different cytoplasmic isoforms of hsp70 proteins, namely, the constitutive hsc73 and the inducible hsp72 isoforms, we found that one isoform related to hsc73 was present in Euglena gracilis. This hsc73-like protein is expressed with a higher rate of synthesis in cells growing under heat shock than in control cells. Moreover, in cadmium-resistant cells, cultured at normal growth temperature, the rate of synthesis of this protein is constitutively increased. These results indicate that a heat-shock protein related to hsc73 is present in an ancestral eukaryote, Euglena gracilis, and that this protein may be constitutive and stress inducible as well.     

Exposure of developing oligodendrocytes to cadmium causes HSP72 induction, free radical generation, reduction in glutathione levels, and cell death

Primary cultures of oligodendrocytes were used to study the toxic effects of cadmium chloride. Cell viability was evaluated by the mitochondrial dehydrogenase activity and confirmed by propidium iodide (PI) fluorescence staining. The expression of the 72 kDa stress protein, HSP72, was assayed by Western blot analysis. The results showed that Cd(2+)-induced toxicity was dependent on the time and dose of exposure, as well as on the developmental stage of the cultures. Oligodendrocyte progenitors were more vulnerable to Cd(2+) toxicity than were mature oligodendrocytes. Mature oligodendrocytes accumulated relatively higher levels of Cd(2+) than did progenitors, as determined by (109)CdCl(2) uptake; treatment with the metal ion caused a more pronounced reduction in intracellular glutathione levels and significantly higher free radical accumulation in progenitors. The latter could explain the observed differences in Cd(2+) susceptibility. HSP72 protein expression was increased both in progenitors and in mature cells exposed to Cd(2+). Pretreatment with N-acetylcysteine, a thiocompound with antioxidant activity and a precursor of glutathione, prevented Cd(2+)-induced (i) reduction in glutathione levels and (ii) induction of HSP72 and diminished (i) Cd(2+) uptake and (ii) Cd(2+)-evoked cell death. In contrast, buthionine sulfoximine, an inhibitor of gamma-glutamyl-cysteine synthetase, depleted glutathione, and potentiated the toxic effect of Cd(2+). These results strongly suggest that Cd(2+)-induced cytotoxicity in oligodendrocytes is mediated by reactive oxygen species and is modulated by glutathione levels.     

Characterization of an aldehyde dehydrogenase from Euglena gracilis

The free-living protist Euglena gracilis showed an enhanced growth when cultured in the dark with high concentrations of ethanol as carbon source. In a medium containing glutamate/malate plus 1% ethanol, E. gracilis reached a density of 3 x 10(7) cells/ml after 100 h of culture, which was 5 times higher than that attained with glutamate/malate or ethanol separately. This observation suggested the involvement of a highly active aldehyde dehydrogenase in the metabolism of ethanol. Purification of the E. gracilis aldehyde dehydrogenase from the mitochondrial fraction by affinity chromatography yielded an enrichment of 34 times and recovery of 33% of the total mitochondrial activity. SDS-PAGE and molecular exclusion chromatography revealed a native tetrameric protein of 160 kDa. Kinetic analysis showed Km values of 5 and 50 microM for propionaldehyde and NAD(+), respectively, and a Vm value of 1,300 nmol (min x mg protein)(-1). NAD(+) and NADH stimulated the esterase activity of the purified aldehyde dehydrogenase. The present data indicated that the E. gracilis aldehyde dehydrogenase has kinetic and structural properties similar to those of human aldehyde dehydrogenases class 1 and 2.     

Changes in phytochelatins and their biosynthetic intermediates in red spruce (<Emphasis Type="Italic">Picea rubens </Emphasis>Sarg.) cell suspension cultures under cadmium and zinc stress

Cell suspension cultures of red spruce (Picea rubens Sarg.) were selected to study the effects of cadmium (Cd) and zinc (Zn) on phytochelatins (PCs) and related metabolites after 24 h exposure. The PC₂ and its precursor, γ-glutamylcysteine (γ-EC) increased two to fourfold with Cd concentrations ranging from 12.5 to 200 μM as compared to the control. However, Zn-treated cells showed a less than twofold increase in γ-EC and PC₂ levels as compared to the control even at the highest concentration of 800 μM. In addition, unidentified higher chain PCs were also found in both the Cd and Zn treated cells and they increased significantly with increasing concentrations of Cd and Zn. The cellular ratio of PC₂ : Cd or Zn content clearly indicated that Cd (with ratios ranging from 0.131 to 0.546) is a more effective inducer of PC₂ synthesis/accumulation than Zn (with ratios ranging from 0.032 to 0.102) in red spruce cells. A marginal decrease in glutathione (GSH) was observed in both Cd and Zn treated cells. However, the GSH precursor, cysteine, declined twofold with all Cd concentrations while the decrease with Zn was 1.5–2-fold only at the higher treatment concentrations of Zn as compared to control. In addition, changes in other free amino acids, polyamines, and inorganic ions were also studied. These results suggest that PCs and their biosynthetic intermediates play a significant role in red spruce cells protecting against Cd and Zn toxicity.     

Cadmium-induced synthesis of HSP70 and a role of glutathione in Euglena gracilis

The effect of cadmium-induced oxidative stress, with or without glutathione supplementation, was investigated in the single cell eukaryotic phytoflagellate, Euglena gracilis strains Z and its achlorophyllous mutant SMZ as experimental models. Both these strains actively synthesize thiols to prevent or resist cadmium toxicity. The content of glutathione, as a representative antioxidant, was also examined in both strains. Exposure to cadmium induced heat-shock protein 70 (HSP70) synthesis in both strains of E. gracilis. Glutathione supplementation also induced HSP70. Overall, these results indicate that glutathione was closely linked to the induction of stress-related proteins. The sensitivity to cadmium-stress was higher in strain Z than SMZ. The results suggest that chloroplasts may have a role in the regulation of HSP70 expression. The relationship between HSP70 and GSH levels is still far from understood, and further research may shed light upon their up-regulation in the presence of Cd.     

Overexpression of cysteine dioxygenase reduces intracellular cysteine and glutathione pools in HepG2/C3A cells

Cysteine levels are carefully regulated in mammals to balance metabolic needs against the potential for cytotoxicity. It has been postulated that one of the major regulators of intracellular cysteine levels in mammals is cysteine dioxygenase (CDO). Hepatic expression of this catabolic enzyme increases dramatically in response to increased cysteine availability and may therefore be part of a homeostatic response to shunt excess toxic cysteine to more benign metabolites such as sulfate or taurine. Direct experimental evidence, however, is lacking to support the hypothesis that CDO is capable of altering steady-state intracellular cysteine levels. In this study, we expressed either the wild-type (WT) or a catalytically inactivated mutant (H86A) isoform of CDO in HepG2/C3A cells (which do not express endogenous CDO protein) and cultured them in different concentrations of extracellular cysteine. WT CDO, but not H86A CDO, was capable of reducing intracellular cysteine levels in cells incubated in physiologically relevant concentrations of cysteine. WT CDO also decreased the glutathione pool and potentiated the toxicity of CdCl(2). These results demonstrate that CDO is capable of altering intracellular cysteine levels as well as glutathione levels.     

Properties of Partially Purified Malate Synthase from Euglena gracilis

SYNOPSIS. Properties of partially purified malate synthase from Euglena gracilis were characterized. The pH optimum is 7.0 and the temperature optimum about 30 C; the activation energy is 12,000 calories. A Km of 4 × 10⁻⁵ M was found for both reactants, glyoxylate and acetyl-CoA. The reaction is partially inhibited by a number of normal metabolites, suggesting allosteric control; glycolate is severely inhibitory. The enzyme is not active in cells grown with phototrophic nutrition, but is found in all heterotrophic cells grown on a wide range of carbon sources; the specific activity is greatly dependent on carbon source. High rates of oxygen consumption are usually, but not always, correlated with high enzyme levels.     

Studies on the Respiratory Physiology of Euglena gracilis Cultured on Acetate or Glucose

SYNOPSIS. The glyoxylate cycle operates at a high level in Euglena gracilis when acetate is the only carbon source, and at a low level when glucose is the only carbon source, as indicated by activities of malate synthase. Altho glucose causes a moderate repression of some of the enzymes of the glyoxylate cycle, it neither represses nor inhibits malate synthase. The specific activity of the malic enzyme was about 5-fold greater in acetate-grown Euglena than in glucose-grown cells, but the absolute rate of CO₂ fixation was about twice as great in cells grown on glucose. The respiratory quotient was unity regardless of substrate.     

Chlorophyll Formation in Euglena gracilis var. bacillaris: Effect of Enzyme Inhibitors

SYNOPSIS. The synthesis of chlorophylls in non-proliferating dark-grown etiolated cells of Euglena gracilis var. bacillaris is markedly inhibited by the enzyme inhibitors dinitrophenol (DNP), p -chloromercuribenzoate (pCMB) and sodium fluoride. The pCMB inhibition of greening is reversed to a great extent by glutathione but not by cysteine. The inhibitory effect of fluoride is reversed by both succinate as well as Mg⁺⁺ during illumination. Fluoride also inhibits the photosynthetic growth of the euglenas; this inhibition is competitively reversed by succinate.     

Characterization and physiological function of glutathione reductase in Euglena gracilis z.

The purified glutathione reductase was homogeneous on polyacrylamide-gel electrophoresis. It had an Mr of 79,000 and consisted of two subunits with a Mr of 40,000. The activity was maximum at pH 8.2 and 52 degrees C. It was specific for NADPH but not for NADH as the electron donor; the reverse reaction was not observed. The Km values for NADPH and GSSG were 14 and 55 microM respectively. The enzyme activity was markedly inhibited by thiol inhibitors and metal ions such as Hg2+, Cu2+ and Zn2+. Euglena cells contained total glutathione at millimolar concentration. GSH constituted more than 80% of total glutathione in Euglena under various growth conditions. Glutathione reductase was located solely in cytosol, as were L-ascorbate peroxidase and dehydroascorbate reductase, which constitute the oxidation-reduction cycle of L-ascorbate [Shigeoka et al. (1980) Biochem. J. 186, 377-380]. These results indicate that glutathione reductase functions to maintain glutathione in the reduced form and to accelerate the oxidation-reduction of L-ascorbate, which scavenges peroxides generated in Euglena cells.     

Amelioration of D-galactosamine-induced acute liver injury in rats by dietary supplementation with betaine derived from sugar beet molasses

The effects of betaine supplementation on D-galactosamine-induced liver injury were examined in terms of hepatic and serum enzyme activities and of the levels of glutathione and betaine-derived intermediates. The rats induced with liver injury showed marked increases in serum enzyme activity, but those receiving dietary supplementation of 1% betaine showed enzyme activity levels similar to a control group without liver injury. Administration of betaine also increased both hepatic and serum glutathione levels, even following D-galactosamine injection. The activity of glutathione-related enzymes was markedly decreased following injection of D-galactosamine, but remained comparable to that of the control group in rats receiving 1% betaine. The concentrations of hepatic S-adenosyl methionine and cysteine showed similar trends to that observed for hepatic glutathione levels. These results indicate that 1% betaine has a hepatoprotective effect by increasing hepatic and serum glutathione levels along with glutathione-related enzyme activities in rats.     

The Alternative Respiratory Pathway of Euglena Mitochondria

Mitochondria, isolated from heterotrophic Euglena gracilis , have cyanide-resistant alternative oxidase (AOX) in their respiratory chain. Cells cultured under a variety of oxidative stress conditions (exposure to cyanide, cold, or H2O2) increased the AOX capacity in mitochondria and cells, although it was significant only under cold stress; AOX sensitivity to inhibitors was also increased by cold and cyanide stress. The value of AOX maximal activity reached 50% of total respiration below 20 degrees C, whereas AOX full activity was only 10-30% of total respiration above 20 degrees C. The optimum pH for AOX activity was 6.5 and for the cytochrome pathway was 7.3. GMP, AMP, pyruvate, or DTT did not alter AOX activity. The reduction level of the quinone pool was higher in mitochondria from cold-stressed than from control cells; furthermore, the content of reduced glutathione was lower in cold-stressed cells. Growth in the presence of an AOX inhibitor was not affected in control cells, whereas in cold-stressed cells, growth was diminished by 50%. Cyanide diminished growth in control cells by 50%, but in cold-stressed cells this inhibitor was ineffective. The data suggest that AOX activity is part of the cellular response to oxidative stress in Euglena .     

Initiation of caspase-independent death in mouse mesangial cells by Cd2+: involvement of p38 kinase and CaMK-II

Cadmium (Cd) is a toxic metal with multiple effects on cell signaling and cell death. We studied the effects of Cd(2+) on quiescent mouse mesangial cells in serum-free conditions. Cadmium induces cell death over 6 h through annexin V+ states without or with causing uptake of propidium iodide, termed apoptotic and apoptosis-like death, respectively. Little or no necrosis is observed, and cell death is caspase-independent and associated with nuclear translocation of the apoptosis-inducing factor, AIF. We previously showed that Cd(2+) increased phosphorylation of Erk and CaMK-II, and CaMK-II activation increased cell death in an Erk-independent manner. Here we demonstrate that Cd(2+) increases Jnk and p38 kinase phosphorylation, and inhibition of p38-but not of Jnk-increases cell viability by suppressing apoptosis in preference to apoptosis-like death. Neither p38 kinase nor CaMK-II inhibition protects against a decrease in mitochondrial membrane potential, psi, indicating that kinase-mediated death is either independent of, or involves events downstream of a mitochondrial pathway. However, both the antioxidant N-acetyl cysteine (NAC) and the mitochondrial membrane-stabilizing agent cyclosporine A (CsA) partially preserve psi, suppress activation of p38 kinase, and partially protect the cells from Cd(2+)-induced death. Whereas the effect of CsA is on apoptosis, NAC acts on apoptosis-like death. Inhibition of glutathione synthesis exacerbates a Cd(2+)-dependent increase in cellular peroxides and favors apoptosis-like death over apoptosis. The caspase-independence of these modes of cell death is not due to an absence of this machinery in the mesangial cells: when they are exposed to Cd(2+) for longer periods in the presence of serum, procaspase-3 and PARP are cleaved and caspase inhibition is protective. We conclude that Cd(2+) can kill mesangial cells by multiple pathways, including caspase-dependent and -independent apoptotic and apoptosis-like death. Necrosis is not prominent. Activation of p38 kinase and of CaMK-II by Cd(2+) are associated with caspase-independent apoptosis that is not dependent on mitochondrial destabilization.     

Involvement of glutathione and glutathione-related enzymes in the protection of normal and trisomic human fibroblasts against daunorubicin

We measured the glutathione content, and the activity of glutathione-related enzymes and DT-diaphorase in cultured normal (cell line: S-126) and trisomic (cell lines: S-158, S-240) human fibroblasts exposed to daunorubicin (DNR). Determination of reduced and total glutathione levels, and measurement of the activity of glutathione peroxidase, glutathione reductase, glutathione-S-transferase and DT-diaphorase were performed spectrophotometrically. Human fibroblasts were exposed to 4 microm DNR for 2 h, and the cells placed in drug-free medium for 6, 12, 24, 48, and 72 h. Cellular levels of GSH and total glutathione decreased following exposure to DNR. However, the ratio of GSH to total glutathione returned to control levels only in trisomic cells. These changes were concomitant with increasing glutathione-S-transferase and glutathione reductase activities. DNR also significantly increased the activity of Se-independent peroxidase and DT-diaphorase in trisomic fibroblasts. Marked increases in the activity of Se-dependent peroxidase and DT-diaphorase alone were seen in normal cells. The results provide the first evidence that DNR can induce alterations in the level of glutathione and glutathione-dependent enzymes in trisomic fibroblasts as compared to normal cells, which may provide additional protection against daunorubicin-induced oxidative stress in trisomic fibroblasts.     

Time-course development of the Cd<Superscript>2+</Superscript> hyper-accumulating phenotype in <Emphasis Type="Italic">Euglena gracilis</Emphasis>

To determine the onset of the Cd²⁺-hyperaccumulating phenotype in Euglena gracilis, induced by Hg²⁺ pretreatment (Avilés et al. in Arch Microbiol 180:1–10, 2003), the changes in cellular growth, Cd²⁺ uptake, and intracellular contents of sulfide, cysteine, γ-glutamylcysteine, glutathione and phytochelatins during the progress of the culture were analyzed. In cells exposed to 0.2 mM CdCl₂, the Cd²⁺-hyperaccumulating phenotype was apparent only after 48 h of culture, as indicated by the significant increase in cell growth and higher internal contents of sulfide and thiol-compounds, along with a higher γ-glutamylcysteine synthetase activity. However, the stiochiometry of thiol-compounds/Cd²⁺ accumulated was similar for both control and Hg²⁺-pretreated cells. Moreover, the value for this ratio was 2.1 or lower after 48-h culture, which does not suffice to fully inactivate Cd²⁺. It is concluded that, although the glutathione and phytochelatin synthesis pathway is involved in the development of the Cd²⁺-hyperaccumulating phenotype in E. gracilis, apparently other pathways and sub-cellular mechanisms are also involved. These may be an increase in other Cd²⁺ chelating molecules such as di- and tricarboxylic acids, phosphate and polyphosphates, as well as Cd²⁺ compartmentation into organelles. César Avilés: In memoriam.