Cu-induced Oxidative Stress Research Articles

Synthesis, Characterization, and in Vitro Anticancer Activity of Copper and Zinc Bis(Thiosemicarbazone) Complexes.

A series of eight bis(thiosemicarbazone) ligands and 16 of their respective copper(II) and zinc(II) complexes containing a combination of hydrogen, methyl, pyridyl, phenyl, and/or ethyl substituents at the diimine position of the ligand backbone were synthesized and characterized. The objective of this study was to identify the structure-activity relationships within a series of analogues with different substituents at the diimine position of the backbone and at the terminal N atom. The Cu(II) complexes Cu(GTSM2), Cu(GTSCM), Cu(PyTSM2), Cu(EMTSM2) and Cu(PGTSM2) demonstrated a distorted square planar geometry, while the Zn(II) complexes Zn(ATSM2)(DMSO), Zn(PyTSM2)(DMSO), and Zn(PGTSM2)(H2O) formed a distorted square pyramidal geometry. Cyclic voltammetry showed that the Cu(II) complexes display quasi-reversible electrochemistry. Of the agents, Cu(II) glyoxal bis(4,4-dimethyl-3-thiosemicarbazone) [Cu(GTSM2)] and Cu(II) diacetyl bis(4,4-dimethyl-3-thiosemicarbazone) [Cu(ATSM2)] demonstrated the greatest antiproliferative activity against tumor cells. Substitutions at the diimine position and at the terminal N atom with hydrophobic moieties markedly decreased their antiproliferative activity. Complexation of the bis(thiosemicarbazones) with Zn(II) generally decreased their antiproliferative activity, suggesting the Zn(II) complex did not act as a chaperone to deliver the ligand intracellularly, in contrast to similar bis(thiosemicarbazone) cobalt(III) complexes [King et al. Inorg. Chem. 2017, 56, 6609-6623]. However, five of the eight bis(thiosemicarbazone) Cu(II) complexes maintained or increased their antiproliferative activity, relative to the ligand alone, and a mechanism of Cu-induced oxidative stress is suggested. Surprisingly, relative to normoxic growth conditions, hypoxia that is found in the tumor microenvironment decreased the antiproliferative efficacy of most bis(thiosemicarbazones) and their copper complexes. This was independent of the potential hypoxia-selectivity mediated by Cu(II/I) redox potentials. These results provide structure-activity relationships useful for the rational design of bis(thiosemicarbazone) anticancer agents.

Acetylcholinesterase and Aβ Aggregation Inhibition by Heterometallic Ruthenium(II)-Platinum(II) Polypyridyl Complexes.

Two heteronuclear ruthenium(II)-platinum(II) complexes [Ru(bpy)2(BPIMBp)PtCl2]2+ (3) and [Ru(phen)2(BPIMBp)PtCl2]2+ (4), where bpy = 2,2'-bipyridine, phen = 1,10-phenanthroline, and BPIMBp = 1,4'-bis[(2-pyridin-2-yl)-1H-imidazol-1-ylmethyl]-1,1'-biphenyl, have been designed and synthesized from their mononuclear precursors [Ru(bpy)2(BPIMBp)]2+ (1) and [Ru(phen)2(BPIMBp)]2+ (2) as multitarget molecules for Alzheimer's disease (AD). The inclusion of the cis-PtCl2 moiety facilitates the covalent interaction of Ru(II) polypyridyl complexes with amyloid β (Aβ) peptide. These multifunctional complexes act as inhibitors of acetylcholinesterase (AChE), Aβ aggregation, and Cu-induced oxidative stress and protect neuronal cells against Aβ-toxicity. The study highlights the design of metal based anti-Alzheimer's disease (AD) systems.

NaCl protects against Cd and Cu-induced toxicity in the halophyte Atriplex halimus

The objective of the present work was to evaluate the extent of Cd- and Cu-induced oxidative stress and the antioxidant response triggered in the halophyte species Atriplex halimus after metallic trace elements exposure. Plants were treated for one month with Cd2+ or Cu2+ (400 µM) in the absence or presence of 200 mM NaCl in the irrigation solution. The interaction between salinity and heavy metal stress was analyzed in relation to plant growth, tissue ion contents (Na+, K+ and Mg2+), oxidative damage and antioxidative metabolism. Data indicate that shoot and root weight significantly decreased as a consequence of Cd2+- or Cu2+-induced stress. Metallic stress leads to unbalanced nutrient uptake by reducing the translocation of K+ and Mg2+ from the root to the shoot. The levels of malondialdehyde increased in root tissue when Cd, and especially Cu, were added to the irrigation solution, indicating that oxidative damage occurred. Results showed that NaCl gave a partial protection against Cd and Cu induced toxicity, although these contaminants had distinct influence on plant physiology. It can be concluded that salinity drastically modified heavy metal absorption and improved plant growth. Salinity also decreased oxidative damage, but differently in plants exposed to Cd or Cu stress.

Histidine Prevents Cu-Induced Oxidative Stress and the Associated Decreases in mRNA from Encoding Tight Junction Proteins in the Intestine of Grass Carp (Ctenopharyngodon idella).

Copper (Cu) is a common heavy metal pollutant in aquatic environments that originates from natural as well as anthropogenic sources. The present study investigated whether Cu causes oxidative damage and induces changes in the expression of genes that encode tight junction (TJ) proteins, cytokines and antioxidant-related genes in the intestine of the grass carp (Ctenopharyngodon idella). We demonstrated that Cu decreases the survival rate of fish and increases oxidative damage as measured by increases in malondialdehyde and protein carbonyl contents. Cu exposure significantly decreased the expression of genes that encode the tight junction proteins, namely, claudin (CLDN)-c, -3 and -15 as well as occludin and zonula occludens-1, in the intestine of fish. In addition, Cu exposure increases the mRNA levels of the pro-inflammatory cytokines, specifically, IL-8, TNF-α and its related signalling factor (nuclear factor kappa B, NF-κB), which was partly correlated to the decreased mRNA levels of NF-κB inhibitor protein (IκB). These changes were associated with Cu-induced oxidative stress detected by corresponding decreases in glutathione (GSH) content, as well as decreases in the copper, zinc-superoxide dismutase (SOD1) and glutathione peroxidase (GPx) activities and mRNA levels, which were associated with the down-regulated antioxidant signalling factor NF-E2-related factor-2 (Nrf2) mRNA levels, and the Kelch-like-ECH-associated protein1 (Keap1) mRNA levels in the intestine of fish. Histidine supplementation in diets (3.7 up to 12.2 g/kg) blocked Cu-induced changes. These results indicated that Cu-induced decreases in intestinal TJ proteins and cytokine mRNA levels might be partially mediated by oxidative stress and are prevented by histidine supplementation in fish diet.

Growth and photosynthetic responses of Lemna minor L. exposed to cadmium in combination with zinc or copper.

Metals have a variety of negative outcomes on plants, essential components of any ecosystem. The effects of CdCl2 (5 μmol L-1), ZnCl2 (25 or 50 μmol L-1), and CuCl2 (2.5 or 5 μmol L-1) and combinations of CdCl2 with either ZnCl2 or CuCl2 on the growth, photosynthetic pigments, and photosystem II (PSII) efficiency of duckweed (Lemna minor L.) were investigated. All of the treatments caused growth inhibition and remarkable metal accumulation in plant tissue after 4 and 7 days. In the combined treatments, the accumulation of each metal applied was lesser in comparison to treatments with single metals. After 4 days, all of the treatments generally diminished chlorophyll a content and decreased the maximum quantum yield (Fv/Fm) and effective quantum yield (ΔF/F'm) of PSII. However, after 7 days of exposure to a combination of Cd and Zn, pigment content and PSII activity recovered to control levels. A higher concentration of Cu (5 μmol L-1) as well as Cd in combination with Cu had a prolonged inhibitory effect on photosynthetic features. Our results suggest that growth inhibition was due to the toxic effect of absolute metal quantity in plant tissue. Zn counteracted Cd uptake, as seen from the recovery of pigment content and PSII efficiency in plants exposed for 7 days to the Cd and Zn combination. Cu-induced oxidative stress led to a prolonged inhibitory effect in plants treated both with a higher concentration of Cu (5 μmol L-1) and simultaneously with Cd and Cu. Our findings could contribute to general knowledge on anthropogenic and environmental contaminants that endanger plant communities and significantly disrupt the sensitive balance of an ecosystem by influencing photosynthetic mechanisms.

Oxidative stress and metabolic responses to copper in freshwater- and seawater-acclimated killifish, Fundulusheteroclitus

In freshwater (FW), many of the main mechanisms of copper (Cu) toxicity have been characterized; however, toxicity mechanisms in seawater (SW) are less well understood. We investigated the effects of salinity on Cu-induced oxidative stress and metabolic responses in adult killifish, Fundulus heteroclitus. We exposed FW and SW-acclimated killifish to either low Cu (LC, 50μg/L) or high Cu (HC, 200μg/L) for 96h and compared them to controls (CTRL) under the same salinities without added Cu. Cu exerted minimal influence on tissue ion levels in either FW or SW. Salinity generally protected against Cu bioaccumulation in the gills and liver, but not in the carcass. Hematocrit (Hct) and hemoglobin (Hb) levels were increased by LC and HC in both FW and SW, and blood lactate was reduced in FW-killifish exposed to LC and HC. Rates of oxygen consumption were similar across treatments. Salinity reduced Cu load in gill, liver and intestine at LC but only in the gills at HC. In general, Cu increased gill, liver, and intestine catalase (CAT) activity, while superoxide dismutase (SOD) either decreased or remained unchanged depending on tissue-type. These changes did not directly correlate with levels of protein carbonyls, used as an index of oxidative stress. Cu-induced changes in carbohydrate metabolic enzymes were low across tissues and the effect of salinity was variable. Thus, while salinity clearly protects against Cu bioaccumulation in some tissues, it is unclear whether salinity protects against Cu-induced oxidative stress and metabolic responses.

Pretreatment with salicylic acid and ascorbic acid significantly mitigate oxidative stress induced by copper in cotton genotypes.

Higher uptake and translocation of copper (Cu) into plant tissues can cause serious physiological and biochemical alterations in root and leaf tissues of plants. The present study investigates the ameliorative role of salicylic acid (SA) and ascorbic acid (AsA) against Cu-induced toxicity changes in cotton genotypes (two parental lines (J208, Z905) and their hybrid line (ZD14)). To study the tolerance potential against Cu (100μM) stress, 2-week-old cotton seedlings were pretreated with 100μM either SA or AsA for three days. Elevated Cu concentration in nutrient media increased Cu accumulation in roots and shoots of all the three cotton genotypes studied. Roots were the main Cu storage site, followed by leaves and stems. Increased cellular Cu concentration significantly inhibited the root and shoot development, although leaf growth was more sensitive toward Cu toxicity. Cu-induced oxidative stress to cotton leaves was evident from significantly increased hydrogen peroxide (H2O2) contents and lipid membrane damage. Increasing Cu translocation toward cotton leaves strongly influenced the malondialdehyde (MDA) contents, which, in turn, inhibited biomass production. SA and AsA pretreated cotton seedlings showed better growth under Cu stress. Despite increase in overall Cu uptake, the SA-pretreated seedlings could defy Cu toxicity through inhibited Cu translocation and modification in the activities of antioxidative enzymes. Whereas, tolerance to Cu-induced toxicity in AsA pretreated plants was associated with Cu exclusion from tissues and reduction of the overall Cu uptake. The present study revealed that the alleviatory role of AsA was significantly higher than SA regarding Cu stress in our experimental cotton genotypes. Furthermore, the hybrid cotton genotype (ZD14) performed well followed by J208 and Z905 in the present experimental setup.

Metallomics and NMR-based metabolomics of Chlorella sp. reveal the synergistic role of copper and cadmium in multi-metal toxicity and oxidative stress.

Industrial wastewaters often contain high levels of metal mixtures, in which metal mixtures may have synergistic or antagonistic effects on aquatic organisms. A combination of metallomics and nuclear magnetic resonance spectroscopy (NMR)-based metabolomics was employed to understand the consequences of multi-metal systems (Cu, Cd, Pb) on freshwater microalgae. Morphological characterization, cell viability and chlorophyll a determination of metal-spiked Chlorella sp. suggested synergistic effects of Cu and Cd on growth inhibition and toxicity. While Pb has no apparent effect on Chlorella sp. metabolome, a substantial decrease of sucrose, amino acid content and glycerophospholipid precursors in Cu-spiked microalgae revealed Cu-induced oxidative stress. Addition of Cd to Cu-spiked cultures induced more drastic metabolic perturbations, hence we confirmed that Cu and Cd synergistically influenced photosynthesis inhibition, oxidative stress and membrane degradation. Total elemental analysis revealed a significant decrease in K, and an increase in Na, Mg, Zn and Mn concentrations in Cu-spiked cultures. This indicated that Cu is more toxic to Chlorella sp. as compared to Cd or Pb, and the combination of Cu and Cd has a strong synergistic effect on Chlorella sp. oxidative stress induction. Oxidative stress is confirmed by liquid chromatography tandem mass spectrometry analysis, which demonstrated a drastic decrease in the GSH/GSSG ratio solely in Cu-spiked cultures. Interestingly, we observed Cu-facilitated Cd and Pb bioconcentration in Chlorella sp. The absence of phytochelatins and an increment of extracellular polymeric substances (EPS) yields in Cu-spiked cultures suggested that the mode of bioconcentration of Cd and Pb is through adsorption of free metals onto the algal EPS rather than intracellular chelation to phytochelatins.

Overexpression of alpha-synuclein at non-toxic levels increases dopaminergic cell death induced by copper exposure via modulation of protein degradation pathways

Gene multiplications or point mutations in alpha (α)-synuclein are associated with familial and sporadic Parkinson's disease (PD). An increase in copper (Cu) levels has been reported in the cerebrospinal fluid and blood of PD patients, while occupational exposure to Cu has been suggested to augment the risk to develop PD. We aimed to elucidate the mechanisms by which α-synuclein and Cu regulate dopaminergic cell death. Short-term overexpression of wild type (WT) or mutant A53T α-synuclein had no toxic effect in human dopaminergic cells and primary midbrain cultures, but it exerted a synergistic effect on Cu-induced cell death. Cell death induced by Cu was potentiated by overexpression of the Cu transporter protein 1 (Ctr1) and depletion of intracellular glutathione (GSH) indicating that the toxic effects of Cu are linked to alterations in its intracellular homeostasis. Using the redox sensor roGFP, we demonstrated that Cu-induced oxidative stress was primarily localized in the cytosol and not in the mitochondria. However, α-synuclein overexpression had no effect on Cu-induced oxidative stress. WT or A53T α-synuclein overexpression exacerbated Cu toxicity in dopaminergic and yeast cells in the absence of α-synuclein aggregation. Cu increased autophagic flux and protein ubiquitination. Impairment of autophagy by overexpression of a dominant negative Atg5 form or inhibition of the ubiquitin/proteasome system (UPS) with MG132 enhanced Cu-induced cell death. However, only inhibition of the UPS stimulated the synergistic toxic effects of Cu and α-synuclein overexpression. Our results demonstrate that α-synuclein stimulates Cu toxicity in dopaminergic cells independent from its aggregation via modulation of protein degradation pathways.

Effect of copper−induced oxidative stress on sclerotial differentiation, endogenous antioxidant contents, and antioxidative enzyme activities of Penicillium thomii PT95

Penicillium thomii PT95 strain is able to form abundant orange, sand-shaped sclerotia in which carotenoids accumulate. We have studied the effects of copper (Cu)-induced oxidative stress on sclerotial differentiation, biosynthesis of some endogenous antioxidants, and activities of a number of the antioxidative enzymes of strain PT95. The association between sclerotial biomass, carotenoid, ascorbate and glutathione contents, and the activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductases (GR) were also analyzed in this fungal strain. We found that the oxidative stress induced by Cu was directly dependent on the concentrations of CuSO4 in the media, with higher CuSO4 concentrations resulting in higher oxidative stress. Cu-induced oxidative stress in strain PT95 was characterized by the initiation of lipid peroxidation. Under Cu-induced oxidative stress growth conditions, the initiation of exudates and sclerotia in strain PT95, as well as sclerotial maturation, were advanced by 1−2 days. Cu-induced oxidative stress favored sclerotial differentiation and the biosynthesis of endogenous antioxidants, i.e., carotenoids, ascorbate, and glutathione. Comparison of SOD, CAT, and GR activities at 0 and 100 μg/ml Cu revealed a 1.1-, 1.8-, and 1.2-fold increase, respectively, at the higher Cu concentration; comparison of their activities at 100 and 300 μg/ml Cu revealed a 1.4-, 3.1-, and 2.2-fold decrease, respectively, at the higher Cu concentration. APX activity decreased linearly with increasing CuSO4 concentration. Our results suggest that the ability of the P. thomii PT95 strain to cope with metal stress is related to its ability to trigger an efficient defense against oxidative stress. These findings may contribute to a better understanding of the response mechanisms of sclerotia production in Penicillium strains to metal stress and to better insights into metal–fungi interactions in natural environments.

Effect of copper on pro- and antioxidative reactions in radish (Raphanus sativus L.) in vitro and in vivo

The generation of superoxide radicals, lipid peroxidation (as measured by malone dialdehyde formation) and the activity of selected antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase) were assessed in radish (Raphanus sativus L.), in response to elevated concentrations of copper ions in the culture medium in vitro and in vivo. Experiments were performed on 7-day-old seedlings and 5-week-old calluses grown on media supplemented with CuSO4 in concentrations of 10, 100 and 1000μМ. The exposure to elevated Cu concentrations in the medium significantly reduced both callogenesis and the proliferation of radish calluses in vitro. Cu treatment resulted in the increased generation of the superoxide radical (O2−) in radish seedlings and calluses indicating the occurrence of oxidative stress in radish cells, whereas the level of lipid peroxidation (LPO) remained unchanged. Both in calluses and in radish seedlings in vivo, the relative level of oxidative stress was maximal at micromolar Cu concentrations and became attenuated with increasing Cu concentrations. Stronger oxidative stress occurred in the radish seedlings in vivo, compared with radish calluses in vitro. The observed lower sensitivity of calluses to Cu-induced oxidative stress and their ability to proliferate upon exposure to Cu concentrations of up to 1000μМ demonstrate the potential of in vitro cell-selection to obtain metal-tolerant radish plant lines.

Erratum to: Influence of arbuscular mycorrhizal fungi and copper on growth, accumulation of osmolyte, mineral nutrition and antioxidant enzyme activity of pepper (Capsicum annuum L.)

The effect of arbuscular mycorrhizal (AM) fungi inoculation on pepper (Capsicum annuum L. cv. Zhongjiao 105) plant growth and on some physiological parameters in response to increasing soil Cu concentrations was studied. Treatments consisted of inoculation or not with Glomus mosseae and the addition of Cu to soil at the concentrations of 0 (control), 2 (low), 4 (medium), and 8 (high) mM CuSO(4). AM fungal inoculation decreased Cu concentrations in plant organs and promoted biomass yields as well as the contents of chlorophyll, soluble sugar, total protein, and the concentrations of P, K, Ca, and Mg. Plants grown in high Cu concentration exhibited a Cu-induced proline accumulation and also an increase in total free amino acid contents; however, both were lower in mycorrhizal pepper. Cu-induced oxidative stress by increasing lipid peroxidation rates and the activity of superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase, and AM symbiosis enhanced these antioxidant enzyme activities and decreased oxidative damage to lipids. In conclusion G. mosseae was able to maintain an efficient symbiosis with pepper plants in contaminated Cu soils, improving plant growth under these conditions, which is likely to be due to reduced Cu accumulation in plant tissues, reduced oxidative stress and damage to lipids, or enhanced antioxidant capacity.

RETRACTED ARTICLE: Influence of arbuscular mycorrhizal fungi and copper on growth, accumulation of osmolyte, mineral nutrition and antioxidant enzyme activity of pepper (Capsicum annuum L.)

The effect of arbuscular mycorrhizal (AM) fungi inoculation on pepper (Capsicum annuum L. cv. Zhongjiao 105) plant growth and on some physiological parameters in response to increasing soil Cu concentrations was studied. Treatments consisted of inoculation or not with Glomus mosseae and the addition of Cu to soil at the concentrations of 0 (control), 2 (low), 4 (medium), and 8 (high) mM CuSO(4). AM fungal inoculation decreased Cu concentrations in plant organs and promoted biomass yields as well as the contents of chlorophyll, soluble sugar, total protein, and the concentrations of P, K, Ca, and Mg. Plants grown in high Cu concentration exhibited a Cu-induced proline accumulation and also an increase in total free amino acid contents; however, both were lower in mycorrhizal pepper. Cu-induced oxidative stress by increasing lipid peroxidation rates and the activity of superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase, and AM symbiosis enhanced these antioxidant enzyme activities and decreased oxidative damage to lipids. In conclusion G. mosseae was able to maintain an efficient symbiosis with pepper plants in contaminated Cu soils, improving plant growth under these conditions, which is likely to be due to reduced Cu accumulation in plant tissues, reduced oxidative stress and damage to lipids, or enhanced antioxidant capacity.

Alleviation of Copper-Induced Oxidative Damage in Chlamydomonas reinhardtii by Carbon Monoxide

Carbon monoxide (CO) is an endogenous gaseous molecule in plants and animals. Recent studies have shown that it is one of the most essential cellular components regulating many aspects of plant growth and development. However, whether CO regulates the green algae adaptive response to heavy metal toxicity is unknown. The present study investigated the role of CO in regulating Cu-induced oxidative stress in eukaryotic algae Chlamydomonas reinhardtii. Cells pretreated with 5μM CO for 30min and followed by exposure to 5μM Cu(II) for 4days showed attenuated toxicity. The CO-improved growth of algae was correlated with reduced lipid peroxidation and increased chlorophyll accumulation. The beneficial effect of CO was confirmed by histochemical staining with reactive oxygen species. Further, treatment with 5μM CO increased the activity of catalase with Cu. However, a reduced superoxide dismutase activity was observed in the CO+Cu-treated algae compared to the control (activity of Cu treatment alone). Under the same condition, the activity of ascorbate peroxidase was not significantly changed. These results suggest that CO can play an important role in regulating the response of algae to Cu stress.

Copper-induced oxidative damage and enhanced antioxidant defenses in the root apex of maize cultivars differing in Cu tolerance

The root apex is highly sensitive to many soil-derived stress factors. Copper (Cu), as a Fenton-type metal, may cause severe oxidative damage in plants at toxic concentrations. The aim of this study was to establish whether the apex is the primary site of Cu-induced oxidative stress and if so, whether there is a site-specific change in antioxidant defenses that can contribute to varietal differences in Cu tolerance. For this purposes, the influence of Cu excess on cell integrity and antioxidant defenses was investigated in two maize cultivars differing in Cu tolerance, Cu-tolerant cv. Oropesa and the Cu-sensitive cv. Orense. Three root zones were considered: 0–5 mm from the root apex (including root cap, meristem and transition zone), 5–10 mm (elongation zone) and 10–15 mm (maturation zone). The 24-h exposure to nominally 2 or 5 μM Cu (pCu7 or 6) confirmed the cultivar differences in Cu tolerance. Both cell membrane integrity, especially at the transition zone in the apex, and root elongation were considerably less damaged by elevated Cu in cv. Oropesa than in cv. Orense. Root tips of both cultivars accumulated similar Cu levels (analyzed after desorption of apoplastic Cu), but 5 μM Cu induced a higher increase of SOD activity (EC 1.15.1.1) in the 0–5 mm root tip region in Oropesa than in Orense. We conclude that this apical root tip zone is the most Cu-sensitive root part, but that the better performance of cv. Oropesa is not due to greater exclusion of Cu from the root apex. Further, the local increase of SOD activity in the root apex (0–5 mm) contributed to the maintenance of cell membrane integrity in the Cu-tolerant cv. Oropesa.

Copper-induced response of physiological parameters and antioxidant enzymes in the aquatic macrophyte Potamogeton pusillus

Bioaccumulation and toxicity of copper was evaluated on Potamogeton pusillus L. The effect of copper (5–100 μg L −1) applied for several days was assessed by measuring changes in the chlorophyll's, phaeophytin's, malondialdehyde, electrical conductivity, glutathione peroxidase (GPX), glutathione reductase (GR) and guaiacol peroxidase (POD) activities. Plants accumulated copper with a maximum of 162 μg g −1 dw after 7-days exposure at 100 μg L −1, however most of the metal was accumulated after 1-day exposure. The toxic effect caused by Cu was evident by the reduction of photosynthetic pigments, increase of malondialdehyde and electrical conductivity. P. pusillus shows Cu-induced oxidative stress by modulating antioxidant enzymes like GPX, GR and POD. Antioxidant enzymes activity increased significantly after exposure to 40 μg L −1 during 24 h, followed by a drop at longer times. Thus, P. pusillus is proposed as a good biomonitor for the assessment of metal pollution in aquatic ecosystems.

Regulation of activity and gene expression of antioxidant enzymes in Ulva fasciata Delile (Ulvales, Chlorophyta) in response to excess copper

T.-M. Wu and T.-M. Lee. 2008. Regulation of activity and gene expression of antioxidant enzymes in Ulva fasciata Delile (Ulvales, Chlorophyta) in response to excess copper. Phycologia 47: 346–360. DOI: 10.2216/07-77.1The concentrations of antioxidants and the activities and gene expression of antioxidant enzymes were determined in Ulva fasciata Delile by a 4-day exposure to 0, 5, 10, 20 and 50 µM CuSO4. H2O2 concentrations were increased by 50 µM Cu, and thiobarbituric acid–reacting substance contents increased as Cu ≥20 µM, reflecting that Cu at higher concentrations induced oxidative stress. Ascorbate and glutathione concentrations and their regeneration were not affected by excess Cu. The transcripts and activities of Mn superoxide dismutase (MnSOD; EC 1.15.1.1), FeSOD and glutathione reductase (GR; EC 1.6.4.2) increased as Cu concentrations increased. The transcripts of ascorbate peroxidase (APX; EC 1.11.1.11) increased linearly as Cu concentrations increased from 0 to 50 µM, while its activities increased as Cu concentrations increased from 5 to 20 µM and then remained unchanged at 50 µM. The transcripts of catalase (CAT; EC 1.11.1.6) increased gradually as Cu concentrations increased, while CAT activities were dramatically increased by 10–50 µM Cu. These results demonstrate that the maintenance of antioxidant homeostasis and the induction of activities of antioxidant enzymes via enhanced gene expression are used by U. fasciata to cope with the Cu-induced oxidative stress, but the defense capacity cannot sufficiently alleviate oxidative damage occurring under the condition of higher Cu concentrations.

Copper toxicity in Withania somnifera: Growth and antioxidant enzymes responses of in vitro grown plants

Indian ginseng ( Withania somnifera L. Dunal), a medicinally important plant exposed to different concentrations of CuSO 4 (0, 10, 25, 50, 100 and 200 μM) for 30 d in vitro accumulated high amount of Cu in a concentration manner. Toxic effects of Cu on plants were reflected by the reductions in fresh weight (FW), shoot and root length, chlorophyll and carotenoids contents. Malondialdehyde (MDA), a decomposition product of polyunsaturated fatty acids hydroperoxides, often considered as a suitable biomarker for lipid peroxidation was induced in metal-treated plants compared to the control. This hypothesis is also confirmed by the higher lipoxygenase (LOX) activity, coupled with higher hydrogen peroxide (H 2O 2) and superoxide anions (O 2 − ) content in treated plants. Ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), glutathione- S-transferase (GST) and guaiacol-peroxidase (G-POD) activities of leaves were increased in the presence of Cu when compared to control plants indicated that antioxidant enzymes played an important role in protecting the plant from Cu toxicity. After native polyacrylamide gel electrophoresis (PAGE) analysis, six APX and four G-POD isoforms were detected and significantly induced in metal-treated plants as compared to control plants. The marked decrease in superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GPx) activities seems to reflect its inability for eliminating the reactive oxygen species (ROS) resulting from Cu-induced oxidative stress. On the other hand, total phenolic contents increased with increasing concentration of Cu compared to the control. This study reveals that plants have the ability to grow in Cu polluted areas by altering various physiological changes. Since the level of total phenolic content in metal-treated plants increases, therefore, it is advisable to assess the biological activity of the plants before using for medicinal purposes.

Physiological responses of Matricaria chamomilla to cadmium and copper excess

Physiological responses of Matricaria chamomilla plants exposed to cadmium (Cd) and copper (Cu) excess (3, 60, and 120 microM for 7 days) with special emphasis on phenolic metabolism were studied. Cu at 120 microM reduced chamomile growth, especially in the roots where it was more abundant than Cd. Notwithstanding the low leaf Cu amount (37.5 microg g(-1) DW) in comparison with Cd (237.8 microg g(-1) DW) at 120 microM, it caused reduction of biomass accumulation, F(v)/F(m) ratio and soluble proteins. In combination with high accumulation of phenolics, strong reduction of proteins and high GPX activity in the roots, this supports severe redox Cu properties. In terms of leaf phenylalanine ammonia-lyase (PAL) activity, it seems that Cd had a stimulatory effect during the course of the experiment, whereas Cu was found to stimulate it after 7-day exposure. The opposite trend was visible in the roots, where Cd had a stimulatory effect at high doses but Cu mainly at the highest dose. This supports the assumption of different PAL time dynamics under Cd and Cu excess. A dose of 60 and 120 microM Cu led to 2- and 3-times higher root lignin accumulation while the same Cd doses increased it by 33 and 68%, respectively. A Cu dose of 120 microM can be considered as limiting for chamomile growth under conditions of present research, while resistance to high Cd doses was confirmed. However, PAL and phenolics seemed to play an important role in detoxification of Cd- and Cu-induced oxidative stress.

Oxidative Stress Response in Gill and Liver of Liza saliens, from the Esmoriz-Paramos Coastal Lagoon, Portugal

Tissue-specific responses against oxidative stress and lipid peroxidation were analyzed in wild adult mullet (Liza saliens) caught in the Portuguese coastal lagoon Esmoriz-Paramos. Parameters measured were catalase (CAT), superoxide dismutase (SOD), and glutathione-S-transferase (GST) activities in liver and gill tissues and lipid peroxidation. The enzyme activities were related to gill histopathological alterations, as well as to heavy metals (Cu and Zn) concentrations in these tissues. Gill epithelium of L. saliens showed histological alterations, such as epithelial hyperplasia resulting in lamellar fusion, epithelial lifting, vasodilatation, and lamellar aneurisms, with a prevalence ranging from 62% to 92%. The highest Cu content was found in liver (379 mg x kg(-1)), while the highest Zn content was observed in gill (119 mg x kg(-1)). SOD and CAT activities showed differences between gill and liver. The highest activities found were SOD in gill (10.1 U/mg protein) and CAT in liver (39.2 mmol/min/mg protein). In gill, CAT activity was negatively related to both Cu levels and gill lifting, while a positive relationship was found between SOD activity and fish age. The positive relationship between Cu and CAT activity in liver suggests that an increase in metabolic level is related to Cu-induced oxidative stress. The decrease in gill CAT activity can be due to osmotic stress caused by damaged gill epithelium. CAT activity in liver is an appropriate biomarker of oxidative stress in the Esmoriz-Paramos lagoon.