Biochemical responses to cadmium exposure in Oncorhynchus mykiss erythrocytes.

Cd is known for its carcinogenic effects, however its mechanism of toxicity and in particular its ability to promote oxidative stress is debated. In fact, although it is considered a redox-inactive metal, at high concentration Cd was shown to promote indirectly oxidative stress. In this study we investigated metal accumulation in ex vivo exposed trout (Oncorhynchus mykiss) erythrocytes and Cd dose-dependent effect in terms of RBC viability, cytosolic and mitochondrial ROS levels as well as its effects on mitochondrial membrane depolarization, hemoglobin stability and precipitation. In the concentration range used, Cd did not affect cell viability. However, metal accumulation was associated with an increase in all oxidative indexes evaluated, except mitochondrial superoxide anion production that, on the contrary, was significantly decreased, probably due to a lowered respiration rate associated with interference of Cd with complex I, II and III, as suggested by the observed Cd-dependent mitochondrial membrane depolarization. On the other hand, hemoglobin destabilisation seems to be the major trigger of oxidative stress in this cell type.

Effect of different metals on oxidative state and mitochondrial membrane potential in trout erythrocytes.

Homeostasis of metal ions is critical for life and excessive exposure can promote cellular damage that could be due to oxidative damage. In this context we evaluated the effects of three different elements (copper, zinc and aluminum) on oxidative stress and mitochondrial functionality in nucleated trout erythrocytes (Oncorhynchus mykiss). Flowcytometric measurements using MitoProbe and DCFDA-H2 as fluorescent probes, indicated that redox active copper was able to influence all the biological parameters considered while redox inert, zinc and aluminum, show no significant effects. Toxicity of Al and Zn represent a debated argument and their ability to interact with other endogenous metal ions/metal binding proteins could play a role modulating their cellular toxicity.

DNA damage and repair following In vitro exposure to two different forms of titanium dioxide nanoparticles on trout erythrocyte.

TiO2 has been widely used to promote organic compounds degradation on waste aqueous solution, however, data on TiO2 nanotoxicity to aquatic life are still limited. In this in vitro study, we compare the toxicity of two different families of TiO2 nanoparticles on erythrocytes from Oncorhynchus mykiss trout. The crystal structure of the two TiO2 nanoparticles was analyzed by XRD and the results indicated that one sample is composed of TiO2 in the anatase crystal phase, while the other sample contains a mixture of both the anatase and the rutile forms of TiO2 in a 2:8 ratio. Further characterization of the two families of TiO2 nanoparticles was determined by SEM high resolution images and BET technique. The toxicity results indicate that both TiO2 nanoparticles increase the hemolysis rate in a dose dependent way (1.6, 3.2, 4.8 µg mL(-1) ) but they do not influence superoxide anion production due to NADH addition measured by chemiluminescence. Moreover, TiO2 nanoparticles (4.8 µg mL(-1) ) induce DNA damage and the entity of the damage is independent from the type of TiO2 nanoparticles used. Modified comet assay (Endo III and Fpg) shows that TiO2 oxidizes not only purine but also pyrimidine bases. In our experimental conditions, the exposure to TiO2 nanoparticles does not affect the DNA repair system functionality. The data obtained contribute to better characterize the aqueous environmental risks linked to TiO2 nanoparticles exposure.

Comparative toxicity of CuO nanoparticles and CuSO4 in rainbow trout.

This study compared the toxicity and accumulation of two different Cu compounds, CuO nanoparticles (NPs) and soluble CuSO4, in erythrocytes and different tissues in rainbow trout (Oncorhynchus mykiss). The crystal structure of CuO NP analysed by XRD indicates that the NP are Tenorite, a monoclinic CuO. The in vitro toxicity results indicate that both Cu compounds increase the haemolysis rate in a dose-dependent way, but the effect was reduced treating cells with CuO NP. Moreover, both Cu compounds induce DNA damage and the entity of the damage, similarly to haemolysis, was more marked in cells treated with CuSO4. In vivo results, obtained after intraperitoneal injection, showed that Cu concentrations were significantly higher in gills (p<0.0001), kidney (p=0.007) and liver (p<0.05) of exposed fish with a significant increase in plasma Cu concentration 15h after CuSO4 treatment. Cu concentrations were significantly higher in fish exposed to CuSO4 than CuO in kidney (p<0.05) and gills (p<0.0001). Significant DNA damage with respect to controls was detected only when Cu was injected as CuSO4. The present data could serve to evaluate environmental Cu toxicity in fish depending on Cu speciation.

Diallyl trisulfide-induced prostate cancer cell death is associated with Akt/PKB dephosphorylation mediated by P-p66shc.

PURPOSE: P66Shc, an isoform of adaptor proteins, is known to mediate various signals including those leading to apoptosis or cell proliferation. Previously, we have shown that diallyl trisulfide (DATS)-induced prostate cancer cell death was mediated by increased ROS formation. In this study, we investigated the role of p66Shc protein and its serine 36 phosphorylation in DATS induced decrease in prostate cancer cell viability (PC-3). METHODS: PC-3 prostate cancer cells were used in this study. Stable cell lines expressing p66ShcS36A or an empty vector have been obtained. Cell viability, concentration of ROS, changes in P-p66Shc and P-Akt and DNA damage were determined. RESULTS: We observed that DATS treatment increased p66Shc phosphorylation at serine 36. Importantly, the phosphorylation was abolished by JNK inhibitor SP600125. Cells expressing plasmid-encoded variant of p66ShcS36A showed much higher resistance to DATS-induced cells death. In addition to that, we observed that DATS-induced ROS formation was completely abolished in cells expressing the p66ShcS36A variant. Interestingly, SP600125 proved to prevent DATS-induced Akt inactivation. In order to confirm that the observed effect is related to phosphorylation of p66Shc, we performed experiments on a stable cell line expressing p66ShcS36A. In such cells, DATS-induced Akt dephosphorylation was significantly reduced. On the other hand, hydrogen peroxide induced Akt activation in PC-3 cells, which was abrogated in cells expressing p66ShcS36A. CONCLUSIONS: Our results uncover a novel signaling pathway with p66Shc being indispensable for DATS-induced inactivation of Akt due to hypophosphorylation.

Involvement of different hemoprotein thiol groups of Oncorhynchus mykiss in cadmium toxicity.

BACKGROUND: Cadmium is considered the seventh most toxic heavy metal as per ATSDR ranking but its mechanism of toxicity is debated. Recently, we evaluated the effects of this metal on the erythrocyte of teleost fish (Oncorhynchus mykiss) leading us to hypothesize that the pro-oxidant activity of cadmium is not linked to mitochondria but more likely to haemoglobin. In this context, the main aim of this work was to detect the ability of Cd to induce structural perturbation in haemoproteins that present different structures and thus different functional properties and to identify what sites of interaction are mainly involved. METHODS: The effect of Cd on the structural destabilization of the different haemoproteins was followed spectrophometrically through their precipitation. In addition, the sites of interaction between the different haemoproteins and bivalent cadmium ions were identified by MIB server followed by molecular docking/molecular dynamics simulations both in the dimeric and tetrameric associations. RESULTS: Cadmium does not influence the autoxidation rate of Mb, HbA and trout HbI. However, the presence of this metal accelerates the precipitation process in trout HbIV in a dose-dependent manner. Moreover, the presence of 1-10-50-250-500-1000 µM GSH, a chelating agent, reduces the ability of cadmium to accelerate the denaturation process although it is not able to completely prevent it. In order to explain the experimental results, a computational investigations was carried out to identify the cadmium cation affinity for the studied haemoglobins and myoglobin, both in their dimeric and tetrameric forms. As a result, the highest affinity cadmium binding sites for fish HbIV are located at the interface between tetramer-tetramer association, indicating that the cation can assist supramolecular protein aggregations and induce complex precipitation. For mammalian Hb, Mb and fish HbI computational investigation did not detect any site where Cd could to induce such aggregation, in line with the experimental results. CONCLUSION: The present study provides new information on the mechanisms of toxicity of cadmium by specific interaction with trout O. mykiss haemoglobin component.

Evaluation of anticancer role of a novel ruthenium(II)-based compound compared with NAMI-A and cisplatin in impairing mitochondrial functionality and promoting oxidative stress in triple negative breast cancer models.

Platinum-based compounds are the most widely used anticancer drugs but, their elevated toxicity and chemoresistance has stimulated the study of others, such as ruthenium-based compounds. NAMI-A and UNICAM-1 were tested in vitro, comparing the mechanisms of toxicity, in terms of mitochondrial functionality and cellular oxidative stress. UNICAM-1, showed a clear mitochondrial target and a cytotoxic dose-dependent response thanks to its ability to promote an imbalance of cellular redox status. It impaired directly mitochondrial respiratory chain, promoting mitochondrial superoxide anion production, leading to mitochondrial membrane depolarization. All these aspects, could make UNICAM-1 a valid alternative for chemotherapy treatment of breast cancer.

Improvement of Attention, Executive Functions, and Processing Speed in Elderly Women as a Result of Involvement in the Nordic Walking Training Program and Vitamin D Supplementation.

Research indicates that life satisfaction declines with age, and cognitive abilities are gradually reduced-mainly attentional functioning and cognitive processing speed. Therefore, scientists seek to find protective factors and test possible intervention programs; moderately intensive physical activity stands out as particularly promising. In this context, we evaluated the influence of Nordic Walking training supported by vitamin D supplementation (as this nutrient is especially deficient in older people in Poland) on the cognitive and psychological functioning of elderly women. A total of 52 healthy elderly women took part in a Nordic Walking training program complemented by vitamin D supplementation. Cognitive functioning was assessed with the Trail Making Test and the D2 Test of Attention. Quality of life and severity of depressive symptoms were measured with the Short Form Health Survey and the Beck Depression Inventory 2. Significant improvements in all aspects of cognitive functioning was observed (p = 0.01-0.47). The study also showed a decrease in depressive symptoms (p = 0.026). Physical activity and adequate levels of vitamin D can be the key factors in maintaining self-reliance in old age. Involvement in Nordic Walking training, supported by vitamin D supplementation, can strengthen the cognitive functioning of older people-reflected in higher attentional capabilities, better executive functions, and improved cognitive processing speed.

Hemoglobin system of Sparus aurata: changes in fishes farmed under extreme conditions.

In order to gain more knowledge on the stress responses of gilhead seabream (Sparus aurata) under extreme conditions, this study investigated the functional properties of the hemoglobin system and globin gene expression under hypoxia and low salinity. The oxygen affinity for the two hemoglobin components present inside the S. aurata erythrocyte was practically identical as was the influence of protons and organic phosphates (Root effect). The quantification of S. aurata hemoglobin fractions performed by HPLC and the data on gene expression of globin chains assayed by PCR indicate that under hypoxia and low salinity there is a change in the ratio between the two different hemoglobin components. The result indicating that the distinct hemoglobins present in S. aurata erythrocyte have almost identical functional properties, does not explain the adaptive response (expression change) following exposure of the animal to hypoxia or low salinity on the basis of their function as oxygen transporter. We hypothesize that other parallel biological functions that the hemoglobin molecule is known to display within the erythrocyte are involved in adaptive molecular mechanisms. The autoxidation-reduction cycle of hemoglobin could be involved in the response to particular living conditions.

Oxidative damage in rat erythrocyte membranes following ethanol intake: effect of ethyl pyruvate.

Alcoholic patients and experimental animals exposed to ethanol display biochemical signs of oxidative damage, suggesting a possible role of free radicals in causing some of the toxic effects of alcohol. The ester derivative, ethyl pyruvate (EP) is stable in solution and should function as an antioxidant and energy precursor. In the present study, the effect of ethanol intake on plasma membrane fluidity, lipid oxidation and antioxidant enzyme activities (GPx, CAT and SOD) were first evaluated. Secondly, the consequences of ethyl pyruvate treatment on the physico-chemical properties of erythrocyte plasma membranes were investigated. The results obtained demonstrate that ethanol induces an increase in lipid peroxidation, a reduction of GPx activity and fluidity in the hydrophilic-hydrophobic region of the bilayer, moreover an increase of fluidity in hydrophobic part of the plasma membrane was measured. When rats were treated with ethyl pyruvate a partially protective effect can be observed for the hydrophilic-hydrophobic region tested by Laurdan, while EP cannot restore the DPH anisotropy values to the control values. In summary, our data indicate that treatment with EP can only partially reduce ethanol plasma membrane perturbation. Since this study shows an ethyl pyruvate dose-dependent effect, it is important to consider the amount of EP required to maintain the right level of membrane fluidity and polarity. These results could be interesting in order to investigate if EP, due to its radical scavenging effect, can prevent oxidative damage induced by ethanol intake and can protect against injure related with ethanol intake.

Lutein, zeaxanthin and astaxanthin protect against DNA damage in SK-N-SH human neuroblastoma cells induced by reactive nitrogen species.

The purpose of this study was to evaluate the ability of the predominant carotenoids (lutein and zeaxanthin) of the macular pigment of the human retina, to protect SK-N-SH human neuroblastoma cells against DNA damage induced by different RNOS donors. Although astaxanthin has never been isolated from the human eye, it was included in this study because its structure is very close to that of lutein and zeaxanthin and because it affords protection from UV-light. DNA damage was induced by GSNO-MEE, a nitric oxide donor, by Na(2)N(2)O(3), a nitroxyl anion donor and by SIN-1, a peroxynitrite-generating agent. DNA damage was assessed using the comet assay, a rapid and sensitive single cell gel electrophoresis technique able to detect primary DNA damage in individual cells. The tail moment parameter was used as an index of DNA damage. The values of tail moment increased in all the samples incubated with the RNOS donors, indicating DNA impairment. Data obtained show that the ability of zeaxanthin, lutein, and astaxanthin to reduce the DNA damage depends on the type of RNOS donor and the carotenoid concentration used. All the carotenoids studied were capable of protecting against DNA damage in neuroblastoma cells when the cells were exposed to GSNO-MEE. However, a different behaviour was present when the other two RNOS donors were used. The presence of a carotenoid alone (without an RNOS donor) did not cause DNA damage. Spectrophotometric studies showed that the order with which tested carotenoids reacted with RNOS was not always in agreement with the DNA protection results. The data from this study provides additional information on the activities of the macular pigment carotenoids of the human retina.

Flunisolide attenuates nitric oxide-induced DNA damage in rat trachea epithelial cells.

In asthma the bronchial epithelium is highly abnormal, with various structural changes. As a consequence, the epithelium becomes an important source of inflammatory mediators that contribute to the ongoing inflammation and remodeling responses occurring in asthma. Compared with normal individuals, the fraction of exhaled nitric oxide (NO) is elevated in patients with asthma, and these levels have been shown to vary with disease activity. Thus, in asthma, epithelial cells may be exposed to large amounts of NO. Increased NO production is associated with the formation of various nitrosating species capable of promoting DNA damage. In this study we investigated the effect of NO on DNA of rat trachea epithelial cells in the presence or absence of flunisolide. Rat airway epithelial cells were prepared and incubated with the NO donor S-nitroso-L-glutathione monoethyl ester (GSNO-MEE). DNA damage was evaluated using single cell gel electrophoresis 'comet assay.' The parameters used as an index of DNA damage were tail length, tail intensity, and tail moment. Results of our study demonstrated that NO induced significant DNA damage in rat airway epithelial cells. Flunisolide in amounts of 11-110 mumol/L significantly reduced all the considered parameters indicating DNA damage. These data indicate that flunisolide may protect epithelial cells from the NO-mediated DNA damage. NO overproduction could contribute to epithelial injury in asthma, and flunisolide seems to attenuate this damage.

Influence of astaxanthin, zeaxanthin and lutein on DNA damage and repair in UVA-irradiated cells.

In order to gain more knowledge about the antioxidant role of the predominant carotenoids (lutein and zeaxanthin) of the human retina, this study investigated their antioxidant activity and capacity. Astaxanthin was also studied, because its structure is very close to that of lutein and zeaxanthin. The antioxidant activity of these molecules was evaluated using chemiluminescence techniques, with lucigenin and luminol as chemiluminogenic probes for the superoxide radical and hydrogen peroxide, respectively. It was found that all three carotenoids have similar superoxide-scavenging activity. The effect on the reduction of H(2)O(2)-luminol chemiluminescence was present in the following order, zeaxanthin>astaxanthinlutein. Possible antioxidant capacity of these three compounds was sought using a biological system consisting of SK.N.SH human neuroblastoma and rat trachea epithelial cells subjected to oxidative stress from exposure to UVA radiation. In particular, we determined whether these compounds were capable of minimizing DNA damage and influencing the kinetics of DNA repair. DNA damage was assessed using the Comet assay, a rapid and sensitive single-cell gel electrophoresis technique used to detect primary DNA damage in individual cells. Neuroblastoma cells appeared more resistant to oxidative irradiation insult. The presence of carotenoids reduced DNA damage when rat epithelial cells were exposed to UVA radiation for 2min. A different result was obtained in experiments performed on neuroblastoma cells; in this case, the presence of carotenoid during UVA exposition increased the damage. The addition of carotenoids to epithelial cells after 2min of UVA exposition did not seem to improve the kinetics of DNA repair; on the contrary, zeaxanthin (after 60' incubation) and lutein (after 180' incubation) showed a genotoxic effect. The addition of carotenoids to neuroblastoma cells after 30' UVA exposition positively influences the kinetics of DNA repair in the first 15min of incubation. At longer exposition times, while the behaviour measured was not constant, a genotoxic effect was not observed. The data from this study provide additional information on the antioxidant and pro-oxidant activities of the predominant macular pigment carotenoids of the human retina.

Effect of tributyltin on trout blood cells: changes in mitochondrial morphology and functionality.

The aquatic environment is the largest sink for the highly toxic organotin compounds, particularly as one of the main sources is the direct release of organotins from marine antifouling paints. The aim of this study was to investigate the mitochondrial toxicity and proapoptotic activity of tributyltin chloride (TBTC) in teleost leukocytes and nucleated erythrocytes, by means of electron microscopy investigation and mitochondrial membrane potential evaluation, in order to provide an early indicator of aquatic environmental pollution. Erythrocytes and leukocytes were obtained from an inbred strain of rainbow trout (Oncorhynchus mykiss). Transmission electronic micrographs of trout red blood cells (RBC) incubated in the presence of TBTC at 1 and 5 microM for 60 min showed remarkable mitochondrial morphological changes. TBTC-mediated toxicity involved alteration of the cristae ultrastructure and mitochondrial swelling, in a dose-dependent manner. Both erythrocytes and leukocytes displayed a consistent drop in mitochondrial membrane potential following TBTC exposure at concentrations >1 microM. The proapoptotic effect of TBTC on fish blood cells, and involvement of mitochondrial pathways was also investigated by verifying the release of cytochrome c, activation of caspase-3 and the presence of "DNA laddering". Although mitochondrial activity was much more strongly affected in erythrocytes, leukocytes incubated in the presence of TBTC showed the characteristic features of apoptosis after only 1 h of incubation. Longer exposures, up to 12 h, were required to trigger an apoptotic response in erythrocytes.

Pyruvate but not lactate prevents NADH-induced myoglobin oxidation.

In this work, we investigated the influence of NADH on the redox state of myoglobin and the roles of pyruvate and lactate in this process. NADH increased the autoxidation rate of myoglobin. Both a drop in pH and partial deoxygenation markedly stimulated the autoxidation process and the influence of NADH. A correlation between met-Mb formation rate and NADH oxidation rate was always observed. The increased rate of Mb autoxidation caused by NADH was inhibited by catalase and pyruvate but not by l-lactate. The antioxidant activity versus H2O2 of both pyruvate and lactate was evidenced by chemiluminescence experiments. The antioxidant activity of lactate disappeared completely in the presence of myoglobin or apo-myoglobin, whereas it was only reduced for pyruvate. These results could be of interest in preventing autoxidation of myoglobin that can contribute to ischemia-reperfusion injury during infarction or high-intensity exercise.

Plasma and mitochondrial membrane perturbation induced by aluminum in human peripheral blood lymphocytes.

Aluminum is a redox-inert element that could induce cell damage via activation of oxidative stress. In this work, the effect of aluminum on different cellular compartments of human peripheral blood lymphocytes was studied. The presence of aluminum induced a lipid peroxidation and physico-chemical modifications at the membrane level. A decrease in fluorescence anisotropy of TMA-DPH and in the polarity of the lipid bilayer with a concomitant shift toward a gel phase was observed, while the pyrene excimerization coefficient (Kex) increased. Flow cytometry measurements, using JC-1, Rhodamine 123 and H2-DCFDA as fluorescent probes, indicated that aluminum induces a slight mitochondrial membrane depolarization that was associated with a moderate increase in reactive oxygen species production. A significative influence on these parameters was measured only at high aluminum concentration.

Ebselen prevents mitochondrial ageing due to oxidative stress: in vitro study of fish erythrocytes.

Nucleated trout erythrocytes under oxidative stress suffer DNA membrane damage and inactivation of glutathione peroxidase. In addition, oxidative damage increases with the age of the cell. In the present paper, we evaluate the effects of oxidative stress and ageing on mitochondrial functionality by means of transmission electron microscopy and cytofluorimetric determination of mitochondrial membrane potential and intracellular levels of reactive oxygen species. The protective activity of the antioxidant organoselenium compound ebselen, a mimic of glutathione peroxidase, is also evaluated. Ebselen prevents the drastic structural and functional changes in mitochondria in aged RBCs induced by oxidative stress. However, the antioxidant does not prevent swelling of the mitochondria.

Effect of NADH on the redox state of human hemoglobin.

In this work, we report that NADH can increase the autoxidation rate of hemoglobin (HbA) in a pH-dependent fashion. During this process, this cofactor is itself oxidized. The presence of superoxide dismutase (SOD) and/or catalase (CAT) can inhibit this result. At lower pH rates, the effect of NADH on the hemoglobin autoxidation rate is more enhanced; in addition, the rate of NADH oxidation is increased. Our data indicates that the reduced pyridine nucleotide may influence the redox state of human hemoglobin by a mechanism, which probably involves free radical species.

A new method to evaluate spontaneous platelet aggregation in type 2 diabetes by Cellfacts.

BACKGROUND: The alterations in the functional activities of platelets in diabetes produce an increase of spontaneous platelet aggregation (SPA) and release of platelet-derived microparticles. Platelet-derived microparticles are shed from platelets during activation by high shear stress, collagen and certain agonists. Although the physiologic role of microparticles has been difficult to assess, the characterization of their biological activity is of interest in view of a possible role in hemostasis and coagulation and their reported involvement in thrombotic disease. METHODS: We propose a new, simple method to evaluate spontaneous platelet aggregation and release of platelet-derived microparticles by the Cellfacts analyser (Microbial System Limited (MSL), Coventry, England) that uses electrical sensing flow impedance determination to detect the size particles and the cells in a conductive fluid. Platelet-rich plasma (PRP) from type 2 diabetes was employed for this study. The importance of platelet-activating factor (PAF) on spontaneous platelet aggregation was evaluated and the effect of vitamin E and WEB 2086-BS, an antagonist of platelet-activating factor, was measured. RESULTS AND CONCLUSIONS: Data presented show that Cellfacts could be an easy and fast instrument to check the state of platelets in patients with alterations in the functionality of platelets, and to follow the effect of pharmacological therapy on spontaneous platelet aggregation and the release of platelet-derived microparticles.

Lymphocyte DNA alteration by sub-chronic ethanol intake in alcohol-preferring rats.

BACKGROUND: Excessive alcohol consumption has been correlated with a higher susceptibility to infections among humans. Chromosome aberrations and other parameters have been suggested as useful biomarkers in assessing genetic damage due to ethanol intake. METHODS: Genetically selected alcohol-preferring rats were given water, 10% ethanol and water or 10% ethanol alone for 3 months as fluid to drink. Food was available ad libitum for the entire period. At the end of the sub-chronic treatment their blood and liver were collected. All blood cells were counted and both lymphocytes and hepatocytes of all three groups were tested with the Comet assay to determine whether any DNA damage had occurred. RESULTS: Only lymphocytes showed DNA damage, with differences among groups. The group that had only ethanol to drink showed greater lymphocyte DNA damage than the ethanol/water and water alone groups. On the other hand, hepatocyte DNA did not show any signs of damage. CONCLUSIONS: Ten weeks of sub-chronic ethanol treatment produces small but significant damage to lymphocytes but not to hepatocytes, a result which confirms the observations of previous authors, and extends them even to a strain of rats genetically selected for high ethanol intake.