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Heavy metals remain a major pollutant in waters near mining sites. Water pollution is a current, longterm problem that affects plants and organisms that live in these water systems, and the effect is very harmful not only for individual species and populations, but also for the entire biological community. This study analyzed the potential of the microalga Chlorella sorokiniana in the adsorption of four selected metals: Fe, Cu, Zn and Ni, all present in high amounts in Lake Robule. This pilot study was conducted to evaluate the potential of these microalgae for possible use in future bioremediation treatment of these water. A laboratory study of metal accumulation in lake water samples lasted for 7 days, after which a total decrease in metal concentration was observed, namely Fe ~25%, Cu ~17%, and Zn ~4% on the seventh day. This study confirms the strong potential of microalgae C. sorokiniana to reduce the presence of heavy metals in conditions known for the low pH value of water with a high percentage of Fe and other heavy metals.

Metabolism of metals in microalgae and adaptation to metal excess are of significant environmental importance. We report a three-step mechanism that the green microalga Chlorella sorokiniana activates during the acquisition of and adaptation to manganese (Mn), which is both an essential trace metal and a pollutant of waters. In the early stage, Mn2+ was mainly bound to membrane phospholipids and phosphates in released mucilage. The outer cell wall was reorganized and lipids were accumulated, with a relative increase in lipid saturation. Intracellular redox settings were rapidly altered in the presence of Mn excess, with increased production of reactive oxygen species that resulted in lipid peroxidation and a decrease in the concentration of thiols. In the later stage, Mn2+ was chelated by polyphosphates and accumulated in the cells. The structure of the inner cell wall was modified and the redox milieu established a new balance. Polyphosphates serve as a transient Mn2+ storage ligand, as proposed previously. In the final stage, Mn was stored in multivalent Mn clusters that resemble the structure of the tetramanganese–calcium core of the oxygen-evolving complex. The present findings elucidate the bioinorganic chemistry and metabolism of Mn in microalgae, and may shed new light on water-splitting Mn clusters.

Nickel is utilized by microalgae as a co-factor of urease. On the other hand, this transition metal represents an important pollutant of aquatic ecosystems. The effects of Ni(II) excess on microalgae and the mechanisms of adaptation are poorly understood. Redox processes represent an important component of the mechanisms of interaction of microalgae with transition metals. Pertinent to this, we analyzed redox changes in Chlorella sorokiniana culture that are induced by high levels of Ni(II). The intracellular level of reactive oxygen species (ROS) showed a rapid two-phase increase that took place prior to Ni accumulation in the cell. This was accompanied by oxidation of thiols and drastic deglutathyonilation of proteins. PAM fluorimetry showed that Ni excess induced an increase in the efficiency of photosystem II and promoted electron flow in chloroplasts, which is most likely responsible for ROS rise. In addition, a rising trend in the chlorophyll level was observed. On the other hand, the level of lipid peroxidation and activities of key antioxidative enzymes were not increased, which implies that oxidative stress is not an important player in Ni adaptation/toxicity. After prolonged exposure the efficiency of photosystem II drops, nickel is accumulated in the cells, and new redox balance is established. Our results imply that redox signalling is involved in Ni-induced metabolic activation and that key changes take place in photosynthetic machinery.

METHODS The impact of a set of concentrations of Mn2+ and Ni2+ on growth of C. sorokiniana culture in 3N-BBM+V medium in the early stationary phase was evaluated by changes in optical density at 750 nm and biomass during 7 days treatment. Mucilage release was analyzed using SEM microscopy. Redox settings were analyzed by oxidation-sensitive fluorescent probe and assays for thiols. RESULTS Ni was more toxic than Mn and affected culture growth at lower concentrations. Microalgal cells started releasing mucilage polymers within 1 h of exposure to 1 mM Mn2+, whereas no mucilage was observed even at 24 h of treatment with equimolar Ni2+. The peak of reactive oxygen species production was reached faster for Ni2+ than Mn2+. Mn-induced drops in the concentration of reduced thiols showed a recovery after 1 h and 24 h. Ni2+-induced drop was irreversible. The observed differences between the impact of Mn and Ni may be related to different redox and coordinative properties and to higher capacities of microalgae to sequester Mn in relation to higher quotas than Ni that are required for normal functio

The effects of a fructose-rich diet and chronic stress on copper metabolism in the kidneys are still understudied. We investigated whether fructose and/or chronic unpredictable stress modulate copper metabolism in a way that affects redox homeostasis, thus contributing to progression of metabolic disturbances in the kidney. We determined protein level of copper transporters, chaperones, and cuproenzymes including cytochrome c oxidase, as well as antioxidant enzymes function in the kidneys of male Wistar rats subjected to 20% liquid fructose supplementation and/or chronic stress. Liquid fructose supplementation increased level of copper chaperone of superoxide dismutase and decreased metallothionein level, while rendering the level of copper importer and copper chaperones involved in copper delivery to mitochondria and trans Golgi network unaffected. Stress had no effect on renal copper metabolism. The activity and expression of renal antioxidant enzymes remained unaltered in all experimental groups. In conclusion, fructose, independently of stress, decreased renal copper level, and modulated renal copper metabolism as to preserve vital cellular function including mitochondrial energy production and antioxidative defense, at the expense of intracellular copper storage.

Microalgae show significant biotechnological potential for remediation of wastewaters polluted with metals, including nickel. Ni enters aquatic ecosystems from fuel combustion, smelting, mining, and electroplating endeavors, and represents a particularly important problem for the access to safe drinking water. It is noteworthy that microalgae utilize Ni as a co-factor of urease. Herein we analyzed the impact of high levels of Ni(II) on freshwater microalga Chlorella sorokiniana in the stationary phase of culture growth. In the concentration range 0.5 to 30 mM, Ni induced a drop in cell density and biomass after 7 days of incubation, whereas significant negative impact was present as early as 2 days for 30 mM. High concentrations also induced a decrease in the level of chlorophylls (a and b) in biomass. In contrast, microalgae that were exposed to moderate stress (1 and 2 mM of Ni) appear to increase photosynthetic activity, indicating the activation of some adaptive mechanism. Scanning electron microscopy showed that C. sorokiniana does not release significant amounts of mucilage polymers in response to Ni, in contrast to the response to some other metals 3. Further, Ni at 1 mM induced an increase in the intracellular production of reactive oxygen species, which appears to reach a plateau after 1 h. The concentration of free thiol groups showed a gradual drop during 24 h of incubation with the same Ni concentration. On the other hand, the decrease in the concentration of reduced glutathione that was observed after 1 h was reversible for 1 mM Ni at 24 h. Ni at 5 mM had a more lasting impact on glutathione. It is important to stress out that the concentrations of reduced glutathione and total glutathione showed very similar trends. This implies that free thiols and reduced glutathione may be ‘lost’ due to glutathionylation of proteins and synthesis of phytochelatins. Our results demonstrate that the response of C. sorokiniana to high Ni levels involves multiple compon

Many neutrophilic and acidophilic microalgal species tolerate high metal concentrations and can survive or colonize metal-polluted waters. They show significant biotechnological potential for the remediation and wastewaters processing. On the other hand, negative effects of metal pollution on microalgae may affect the function of aquatic ecosystems because these photosynthetic microorganisms represent the primary producers of O2 and biomass. However, adaptive mechanisms that microalgae employ to detoxify metal excess are largely unknown. Herein we analyzed the response of the freshwater microalga Chlorella sorokiniana to high but non-toxic levels of Mn2+. Manganese is a key metal pollutant, with five possible oxidation forms that can bind to a variety of different ligands. At pH below 7, it is predominantly present in Mn2+ form. Scanning electron microscopy showed that in response to 1 mM Mn2+, C. sorokiniana released mucilage polymers within 1 h. Electron paramagnetic resonance spectroscopy (EPR) showed that the early response involved loose Mn2+ binding to mucilage and/or the cell wall. The amount of loosely bound Mn2+ was significantly decreased after 24 h, whereas biomass showed significant accumulation of Mn, O and P, as determined by energy dispersive X-ray spectrometry, indicating the production of polyphosphates, which may sequester Mn. Further, it was found that the exposure to Mn2+ resulted in rapid and transient decrease of total free glutathione concentration; the drop was observed after 1 h, and the concentration returned to initial values after 24 h. EPR measurements showed a similar trend in the level of reduced thiols. The observed changes can be explained either by the synthesis of phytochelatins – sulfurrich short-chain peptides that sequester metals, or by glutathionylation of proteins. Reduced thiols could not be detected in the extracellular space, indicating that C. sorokiniana did not release thiols in response to high Mn. These results demons

Microalgae can be implemented in the remediation of mining and other metal-rich wastewaters as pioneer species. On the other hand, metals affect aquatic ecosystems through the negative impact on microalgae which are the primary producers of O2 and biomass. Redox processes represent an important component of the mechanisms of interaction of microalgae with transition metals. We analyzed the redox changes in Chlorella sorokiniana culture that are induced by high levels of Mn(II). Mn is the key metal pollutant, with five main oxidation forms that can bind to a variety of different ligands. Mn (1 mM) induced a significant increase in the intracellular production of reactive oxygen species. The boost appears to show two phases – the first is very fast (observed after 15 min), whereas the second starts after 1 h reaching a plateau at 24 h. The concentration of reduced thiols, which represent important targets of oxidation, appears to parallel this trend. Total glutathione concentration shows a drop at 1 h and recovery at 24 h. This implicates that either a glutathionylation of proteins or a synthesis of phytochelatins - sulfur-rich short-chain peptides that sequester metals, takes place early in the response to Mn. Further, FTIR analysis showed that Mn induced a decrease of C=C levels and CH2/CH3 ratio implicating increased lipid peroxidation. Finally, Mn ions that were accumulated in the cells were extracted with nitric oxide and analyzed by cyclic voltammetry. Two redox forms were detected - Mn(II) and Mn(IV). The latter appears to prevail at higher manganese concentrations and longer periods of incubation. These results demonstrate that redox response of C. sorokiniana to high Mn levels involves at least two phases. Initially, Mn(II) enters the cells and induces pro-oxidative changes that are mitigated by glutathione-based antioxidative defense. Later on, redox homeostasis is reestablished with concomitant inactivation of Mn in the more stable redox form.

Introduction Disruption of copper homeostasis and dysfunction of mitochondria have been documented in sclerotic hippocampi (HS) of patients with mesial temporal lobe epilepsy (mTLE). However, a potential link between these pathological changes has not been tackled so far. Herein, we analyzed regional distribution of neuron somata density, copper concentration, and the activity of cytochrome c oxidase (CCO), a component of mitochondrial electron transport chain and copper-containing metalloprotein, in HS. Methods Histochemical staining and laser ablation inductively coupled plasma mass spectrometry were carried out to construct comparable maps of these parameters in coronal sections of hippocampi of 3 mTLE-HS patients and 3 control subjects. Results Copper levels were decreased in all regions of HS with pyramidal neuron somata. CCO activity was significantly reduced in stratum pyramidale (PY) 1 and cornu Ammonis field 4, the two regions with significant reduction in neuron somata density. CCO activity was also lower in layers that contain apical dendrites of pyramidal neurons and mossy fibers. It appears that copper deficiency in PY2 and PY3 comes before CCO activity reduction and neuronal loss. A strong positive correlation was found between neuron density, Cu concentration, and CCO activity. Conclusions Presented results imply that pathological alterations in Cu and energy metabolism could be involved in the development of HS. A limitation of this study was the relatively small number of patients. However, presented results underline copper deficiency as a component of pathological mechanisms of epilepsy and warrant further investigation of cuproproteins and members of copper transport machinery.

Upon release in response to stress, epinephrine (Epi) may interact with labile iron pool in human plasma with potentially important (patho)physiological consequences. We have shown that Epi and Fe3+ build stable 1:1 high-spin bidentate complex at physiological pH, and that Epi does not undergo degradation in the presence of iron. However, the interactions of Epi with the more soluble Fe2+, and the impact of iron on biological activity of Epi are still not known. Herein we showed that Epi and Fe2+ build colorless complex which is stable under anaerobic conditions. In the presence of O2, Epi promoted the oxidation of Fe2+ and the formation of Epi-Fe3+ complex. Cyclic voltammetry showed that mid-point potential of Epi-Fe2+ complex is very low (−582 mV vs. standard hydrogen electrode), which explains catalyzed oxidation of Fe2+. Next, we examined the impact of iron binding on biological performance of Epi using patch clamping in cell culture with constitutive expression of adrenergic receptors. Epi alone evoked an increase of outward currents, whereas Epi in the complex with Fe3+ did not. This implies that the binding of Epi to adrenergic receptors and their activation is prevented by the formation of complex with iron. Pro-oxidative activity of Epi-Fe2+ complex may represent a link between chronic stress and cardiovascular problems. On the other hand, labile iron could serve as a modulator of biological activity of ligands. Such interactions may be important in human pathologies that are related to iron overload or deficiency.

Oxidative status of maternal blood represents an important parameter of pregnancy that is involved in both, regulation of physiological processes and (if significantly altered) development of different pregnancy complications. Inherited thrombophilias represent genetic disorders that increase the risk of thromboembolism in pregnancy. Little is known about the impact of thrombophilia on the oxidative status of maternal blood. In this study, we analyzed oxidative status of blood of 56 women with pregnancies burdened by inherited thrombophilias. The status was established at three different trimesters using biochemical assays and electrochemical measurements, and it was compared to 10 age- and trimester-matching controls. Activities of superoxide dismutase, catalase, and glutathione reductase in the 1(st)and the 2(nd)trimester of thrombophilic pregnancy were lower than controls. Also, there was less oxidation in the plasma, according to higher concentration of reduced thiols and lower oxidation-reduction potential. Therefore, it appears that thrombophilic mothers do not experience oxidative stress in the circulation in the first two trimesters. However, the rise in GPx, GR and SOD activities in the 3(rd)trimester of thrombophilic pregnancy implies that the risk of oxidative stress is increased during the late pregnancy. These results are important for developing antioxidative treatment that could tackle thrombophilia-related pregnancy complications.

Aims: A drop in copper level and the loss of energy homeostasis are both portrayed in mesial temporal lobe epilepsy (mTLE) with hippocampal sclerosis (HS) patients. Cytochrome c oxidase (COX) represents a crossroad of energy and copper metabolism; it is a key component of mitochondrial machinery and contains two copper centers. Our aim here was to examine the link between COX activity and the copper transporting system in HS. COX activity and the levels of mRNA of selected chaperones - COX11, COX17, Sco1 and Sco2 were determined in 13 anatomically distinct hippocampal regions. Methods: Study was performed on seven hippocampal samples, four of which had been acquired during the course of amygdalohippocampectomy treatment of medically intractable epilepsy and three control postmortem samples. Adjacent slices were used for Nissl staining, COX activity assay and mRNA in situ hybridization with autoradiography. Densitometry was performed using ImageJ. Results: Overall COX activity was decreased in HS compared to controls (P = 0.0003). However, 5 regions showed significantly lower COX activity in HS and 8 did not. Subiculum showed slightly higher activity in HS. The levels of mRNA levels were lowered in HS in 6 regions for COX11, 10 regions for COX17, two regions for Sco1 and 11 regions for Sco2. Conclusions: Our findings suggest the loss of energy homeostasis in HS may be related to pathological changes in specific components of copper delivery to COX, and that the impact may vary between different hippocampal regions.

More and more studies are identifying the regulation of metal homeostasis as one of the key points of central nervous system’s well-being. Epilepsy is a particularly interesting neurological condition when viewed in terms of the correlation between the amount of metals and the development of a seizure. This lecture will present contribution of our group to the field of metal biology in epilepsy by mapping brain metals in sclerotic hippocampus resected from drug resistant mesial temporal lobe epilepsy (mTLE) patients as surgical therapeutic approach. Direct insight into this epileptogenic area, by two powerful techniques, optical emission and mass spectrometry, has led us to investigation of copper turnover. Namely, among the examined metals, we found the deficiency of copper in sclerotic hippocampus on two levels: (i) in whole structure (ii) and locally in the areas of neuronal loss, with significant correlation between copper concentration and neuron density. Furthermore, analysis of copper metalloproteins showed: (i) significant increase or decrease in levels of protein that is participating in copper transport into the cell (CTR1) depending on the degree of hippocampal neuronal loss; (ii) and lower activity of an enzyme in which copper is part of the active site, cytochrome c oxidase, in sclerotic hippocampi of patients compared to control tissue. In our further investigations it remained to be determined whether changes in copper concentrations and copper metalloproteins are causal to pathology of mTLE or they represent epiphenomenon.

The monitoring of progression in amyotrophic lateral sclerosis (ALS) relies on clinical outcome measures that take months to interpret, such as revised ALS functional rating scale (ALSFRS-R) score, with no approved biomarkers. A number of clinical studies have documented the involvement of oxidative stress in ALS pathology. Pertinent to this, we propose to evaluate oxidation-reduction potential (ORP) of cerebrospinal fluid (CSF) as a potential indicator of ALS progression. The case-control study included 24 patients with neurological non-neurodegenerative disorders (controls) and 82 ALS patients with different degrees of disease (ALSFRS-R score: 21-47). ORP was significantly higher in ALS patients than controls. It was not dependent on age or gender. A strong negative correlation was found between ORP and ALSFRS-R score for all patients and patients with spinal onset. In other words, ORP increased with ALS progression. No correlation was found for the subset of patients with bulbar onset, most likely because of the physical distance between neurodegenerative loci and the site of CSF collection. These results lead to the hypothesis that ORP of CSF has a potential as monitoring biomarker in ALS, particularly in the cohort of patients with spinal onset. Antioxid. Redox Signal. 00, 000-000.

Mesial temporal lobe epilepsy (mTLE) represents the most common subtype of human focal epilepsies and perhaps the best-characterized disorder of this type 1 . Hippocampal sclerosis (HS) is the most common histopathologic abnormality found in adults with drugresistant mTLE 2 . The histopathologic hallmark of HS is segmental pyramidal cell loss, which can affect any field of the cornu Ammonis (CA1-4). Hippocampal neuronal cell loss is always associated with a severe pattern of astrogliosis 3 . Therewithal, disturbed homeostasis of metals is implicated in the pathology of mTLE-HS. Zinc has been considered to play a major role in epileptogenesis in relation to its involvement in the modulation of excitability and synaptic plasticity 4,5. Further, it has been shown that epileptogenic hippocampi are exposed to oxidative stress and that the development of prooxidative conditions in the CNS usually involves the loss of homeostasis of iron 6,7. Low brain levels of copper and manganese have been reported in patients with Menkes disease and in animal models of epilepsy, and linked to seizure development 8,9. Ristić et al. were the first to conduct a case-control study of total concentration of metals in tissue of human HS, and report lower concentrations of copper and manganese 10.

The loss of metal homeostasis has been implicated in the pathophysiology of mesial temporal lobe epilepsy associated with hippocampal sclerosis (mTLE-HS). Here we applied laser ablation inductively coupled plasma mass spectrometry imaging to establish the spatial distribution of Zn, Fe, Cu and Mn in coronal sections of hippocampi of four patients with drug-resistant mTLE-HS who underwent amygdalohippocampectomy. Detailed maps of the metal concentrations in the different morphological areas/layers were built and analyzed. The highest level of Zn (>20 mu g g(-1)) was found in mossy fiber-rich regions -cornu ammonis field 4 (CA4), gyrus dentatus, and CA3. The distribution of Fe appears to reflect the routes of the main intrahippocampal blood vessels. The highest concentrations of Cu (410 mg g(-1)) and Mn (415 mu g g(-1)) were observed in regions/layers with neuron somata - subiculum, CA4, gyrus dentatus, and stratum pyramidale (SPy) in CA1 and CA2. Alveus and other regions with axons and dendrites generally showed lower levels of Zn, Cu, and Mn. The Cu concentration was decreased in the areas of total neuronal loss in SPy in CA1 (9.73 +/- 0.91 mu g g(-1)), compared to the subiculum (13.32 +/- .29 mu g g(-1); p = 0.043). The Cu and Mn concentrations correlated positively with neuron density in the SPy in CA1 (R = 0.629, p o 0.001; and R = 0.391, p = 0.004). These results provide a deeper insight into hippocampal metabolism of metals, and pave the road for identifying the components of the mechanism of epileptogenesis among Cu and Mn transporters and metalloproteins.

Background: ALS is an oxidative stress-related fastpaced motor neuron disease for which there are no accurate biomarkers of progression. This largely hampers the development of therapeutics. Our aim was to test the applicability of oxidation-reduction potential (ORP) of cerebrospinal fluid (CSF) in ALS progression follow-up. Methods: ORP was measured using RedoxSYS (Aytu BioScience, Inc.) in CSF of 49 ALS patients (mean age 63 1.29 years; range 43-80 years; mean ALSFRSr score 37.47 0.90, range 21-48; m/f = 35/14) and 15 controls (mean age 48.93 3.68 years, range 21-67 years; m/f = 10/5). Results: Pearson correlation coefficient (R) between ALSFRSr score and ORP was 0.27 (p = 0.06). R was higher ( 0.45) when two outlier values were excluded from the calculus. Importantly, ALS patients had significantly higher mean ORP (ALS: 121.83 2.76mV; controls: 111.14 2.94 mV; p = 0.033). The difference was also significant for ORP normalized to age. Of note, ORP is reciprocally proportional to the pro-oxidative settings in biological samples. Discussion: Increased ORP values in ALS patients further confirm the role of oxidative stress in this neurodegenerative disease. ORP might find application as a biomarker for ALS progression/prognosis but further measurements on a larger cohort is warranted.

We have developed a simple one-step 30-min method for fluorescent visualization of proteins in native and sodium dodecyl sulfate polyacrylamide gel electrophoresis (PAGE) gels. The method is based on formation of strong fluorophores via potassium ferricyanide-provoked oxidation of tryptophan (Trp). Following PAGE, gels are soaked in water solution of potassium ferricyanide (100 mM) and NaOH (1 M) and are kept in the dark for 30 min. Gels are then transferred to water and scanned. The sensitivity of the method was slightly lower compared with standard Coomassie Brilliant Blue (CBB) staining. The method can be useful when rapid acquisition of data is of the essence. After preview, gels can be post-stained using the CBB protocol for further analysis. The intensity of fluorescence is dependent on Trp number, so the protocol might find application in the quantification of Trp residues as illustrated here. Importantly, there is room for improvement of the method. Namely, according to excitation-emission matrix analysis of stained protein bands, maximal fluorescence intensity (at 345/460 nm) was 3.5-fold higher compared with the settings that were available on a commercial imager (395/525 nm). As a supplement, we present an upgrade of the previously described method for in-gel detection of non-heme iron-binding proteins that also employs potassium ferricyanide.