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.

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.

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.

High-fat diet-induced met-hemoglobin formation in rats prone (WOKW) or resistant (DA) to the metabolic syndrome: effect of CoQ10 supplementation.

The aim of this study was to evaluate the effects of a high-fat diet (HFD) on oxidative indexes in WistarOttawaKarlsburg W (WOKW) rats used as a model of metabolic syndrome in comparison with Dark Agouti (DA) rats used as a control strain. This syndrome is defined by the occurrence of two or more risk factors including obesity, hypertension, dyslipidemia, and insulin resistance. Forty rats were used in the study and the effect of HFD was evaluated in terms of body weight and both hemoglobin and CoQ oxidative status. Moreover, 16 rats (8 of each strain) were supplemented with 3 mg/100 g b.w. of CoQ10 for 1 month in view of its beneficial properties in cardiovascular disease due to its antioxidant activity in the lipid environment. HFD promoted an increase in body weight, in particular in WOKW males, and in the methemoglobin (met-Hb) index in both strains. Moreover, HFD promoted endogenous CoQ10 oxidation. CoQ10 supplementation was able to efficiently counteract the HFD pro-oxidant effects, preventing met-Hb formation and CoQ oxidation.

Antioxidant and anti-inflammatory activities of extracts from Cassia alata, Eleusine indica, Eremomastax speciosa, Carica papaya and Polyscias fulva medicinal plants collected in Cameroon.

BACKGROUND: The vast majority of the population around the world has always used medicinal plants as first source of health care to fight infectious and non infectious diseases. Most of these medicinal plants may have scientific evidence to be considered in general practice. OBJECTIVE: The aim of this work was to investigate the antioxidant capacities and anti-inflammatory activities of ethanol extracts of leaves of Cassia alata, Eleusine indica, Carica papaya, Eremomastax speciosa and the stem bark of Polyscias fulva, collected in Cameroon. METHODS: Chemiluminescence was used to analyze the antioxidant activities of plant extracts against hydrogen peroxide or superoxide anion. Comet assays were used to analyze the protection against antioxidant-induced DNA damage induced in white blood cells after treating with hydrogen peroxide. Flow cytometry was used to measure γδ T cells proliferation and anti-inflammatory activity of γδ T cells and of immature dendritic cells (imDC) in the presence of different concentrations of plant extracts. RESULTS: Ethanol extracts showed strong antioxidant properties against both hydrogen peroxide and superoxide anion. Cassia alata showed the highest antioxidant activity. The effect of plant extracts on γδ T cells and imDC was evidenced by the dose dependent reduction in TNF-α production in the presence of Cassia alata, Carica papaya, Eremomastax speciosa Eleusine indica, and Polyscias fulva. γδ T cells proliferation was affected to the greatest extent by Polyscias fulva. CONCLUSION: These results clearly show the antioxidant capacity and anti-inflammatory activities of plant extracts collected in Cameroon. These properties of leaves and stem bark extracts may contribute to the value for these plants in traditional medicine and in general medical practice.

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.

In vitro effect of AlCl3 on human erythrocytes: changes in membrane morphology and functionality.

Aluminum belongs to a group of potential toxic elements capable of penetrating the human body. In this paper, the effect of aluminum concentrations on red blood cell membranes using different fluorescent probes able to localize in various parts of the phospholipid bilayer (TMA-DPH, laurdan and pyrene) were studied. Our results confirm that human erythrocytes exposed to aluminum undergo 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, and the pyrene excimerization coefficient (kex) increased. Furthermore, the presence of aluminum induced lipid peroxidation and reduced the activity of erythrocyte antioxidant enzymes (SOD, CAT and GSHPx). Al-induced morphological changes on the erythrocyte membrane surface were monitored using atomic force microscopy. These results provide further information on the target of action of different aluminum amounts.

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.

Molecular cloning and characterization of adult Sparus aurata hemoglobin genes.

Among Teleosts, Sparus aurata occupies a prominent place in the gastronomic and economic fields of the Mediterranean basin and other geographic districts. The knowledge of its molecular structures and functional features, such as hemoglobin, may be helpful to understand the adaptive biochemical mechanisms that allow this fish to live under extreme conditions, including fish farming. In Sparus aurata red blood cells two different alpha and one beta hemoglobin genes have been identified. The alpha1 gene codifies a putative protein of 144 amino acids, the alpha2 gene produces a protein of 143 amino acids, and the beta gene encodes a chain of 148 amino acids. Comparative analysis of various hemoglobins indicates that allosteric regulation can be modified by the substitution of one or a few key residues. The comparison of the percentage sequence differences for alpha and beta chains in fishes indicates that evolutionary relationships between different species may be helpful to understand the mechanisms of their differentiation from other vertebrates. Hemoglobin alpha and beta chains of about 50 teleostean temperate and Antarctic fishes were analyzed to build phylogenetic trees using different algorithms: the neighbor-joining method, the maximum likelihood approach, and the Bayesian inference computation. Sparus aurata alpha chains are positioned in a paraphyletic cluster, which includes the same subunit of Chrysophrys auratus and Seriola quinqueradiata, whereas the beta chain is on an homophyletic branch with that of Chrysophrys auratus. Therefore, the phylogenetic approach suggests that both Sparus aurata hemoglobin alpha genes are paralogues and may have derived from a duplication event.

Oxidative damage in trout erythrocyte in response to "in vitro" copper exposure.

The oxidative action of copper on different trout Oncorhynchus mykiss erythrocyte components was studied. The results indicate that: --cupric ions differently influence the oxidative status of two trout hemoglobin components I and IV (HbI and HbIV) having different structural and functional properties; --reactive oxygen species (ROS) production associated with hemoglobins autoxidation is not influenced by the presence of copper sulfate; --the susceptibility to hemolysis increases in the presence of copper only when the erythrocyte suspension is incubated in air; the effect of copper is almost absent for carbon monoxide-saturated erythrocyte suspensions; --DNA damage due to copper was not observed in our experimental conditions. The data obtained are important for the analysis of the environmental risks produced by copper on fish.

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.

Multiple strategies for O2 transport: from simplicity to complexity.

O(2)carriers (extracellular and intracellular as well as monomeric and multimeric) have evolved over the last billion of years, displaying iron and copper reactive centers; very different O(2)carriers may co-exist in the same organism. Circulating O(2)carriers, faced to the external environment, are responsible for maintaining an adequate delivery of O(2)to tissues and organs almost independently of the environmental O(2)partial pressure. Then, intracellular globins facilitate O(2)transfer to mitochondria sustaining cellular respiration. Here, molecular aspects of multiple strategies evolved for O(2)transport and delivery are examined, from the simplest myoglobin to the most complex giant O(2)carriers and the red blood cell, mostly focusing on the aspects which have been mainly addressed by the so called 'Rome Group'.

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.

Protective effect of ethyl pyruvate on msP rat leukocytes damaged by alcohol intake.

Alcohol consumption for long periods negatively influences physiological functions of many cells, and leads to organ damage. Reactive oxygen and nitrogen species produced by ethanol metabolism cause adverse effects that might be alleviated by simultaneous treatment with various antioxidants. Here, the ability of ethyl pyruvate (EP) to reduce ethanol-induced oxidative stress was evaluated. Chemiluminescence studies show that EP has a higher capacity than pyruvate to scavenge hydrogen peroxide and superoxide anions. In order to evaluate whether EP can exert a protective effect against ethanol, rats were offered 10% ethanol in drinking burettes, containing or not different concentrations of EP (0.3%, 1% and 3%). The comet assay was employed to quantify the alcohol-induced DNA damage in rat lymphocytes. This test is a promising tool for the estimation of DNA damage at the single cell level. A significant protective effect of EP was observed in rat groups treated with this antioxidant, compared with those drinking only ethanol. Since EP has been shown to decrease the expression of numerous pro-inflammatory mediators, the monocyte respiratory burst was evaluated. The activation of monocyte NADPH oxidase by phorbol esters (PMA) showed that superoxide anion production was higher in the ethanol group than in the control group. The presence of EP considerably reduced superoxide anion production. In conclusion, hypotheses on possible mechanisms of action of EP on rat white blood cells are proposed.

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.