Laura A. Geracitano

Behavioral and genoprotective effects of Vaccinium berries intake in mice

Studies have shown that supplementation with berries rich in anthocyanins are effective in reducing oxidative stress associated with aging, and are beneficial in reversing age-related neuronal and behavioral changes. However, there are few reports on other biological activities of these polyphenols, such as genoprotective effects. The present experiments were performed to study the possible effects of 30-day administration of a lyophilized extract of Vaccinium ashei berries on cognitive performance using step-down inhibitory avoidance, open-field habituation and elevated plus-maze tasks, as well as on DNA damage in the hippocampus and cerebral cortex. The present study showed that the extract significantly enhanced long-term memory in the inhibitory avoidance task, induced an increase in the number of crossings during open-field habituation and had an anxiolytic effect in the elevated plus-maze task. Moreover, the extract reduced oxidative DNA damage in brain tissue in vitro. These results suggest that supplementation with V. ashei berries to mice improves performance on memory tasks and has a protective effect on DNA damage, possibly due to the antioxidant activity of polyphenols, including anthocyanins.

Physiological and antioxidant enzyme responses to acute and chronic exposure of Laeonereis acuta (Polychaeta, Nereididae) to copper

Chronic (14 days) and acute (48 h) copper effects on the antioxidant defense system and some physiological variables of Laeonereis acuta (Polychaeta, Nereididae) were evaluated. In both assays, two nominal copper concentrations (chronic: C1=31.25 and C2=62.50 μg/l; acute: A1=250 and A2=500 μg/l) and one control group (Cc and Ac=0 μg/l) were tested. End points analyzed were antioxidant enzyme activity (catalase, CAT; superoxide dismutase, SOD; and glutathione S-transferase, GST), oxygen consumption, metahemoglobin concentration, and lipid peroxidation (LPO). In the chronic assay, CAT activity was significantly higher in worms exposed to both concentrations of copper tested (C1=3.36±0.07 U CAT/mg protein; C2=4.06 0.32 U CAT/mg protein) than in control worms (Cc=2.16±0.39 U CAT/mg protein). SOD activity was also increased in the two copper-exposed groups (C1=16.85±4.22 U SOD/mg protein; C2=38.19±4.31 U SOD/mg protein) than in control group (Cc=3.54±0.46 U SOD/mg protein). However, GST activity was increased only in worms exposed to the higher copper concentration (C2=0.022±9.10−4 U GST/mg protein) when compared to the other groups tested (Cc=0.012±3.10−3 U GST/mg protein; C1=0.016±9.10−4 U GST/mg protein). None of the physiological variables analyzed (oxygen consumption, metahemoglobin concentration, and lipid peroxidation) was affected by chronic copper exposure. In the acute assay, only GST activity was induced in worms exposed to copper. This induction was observed only in the A1 group (0.027±2.10−3 U GST/mg protein) when compared to Ac (0.017±2.10−3 U GST/mg protein) or A2 (0.016±7.10−4 U GST/mg protein) groups. On the other hand, lipid peroxidation was higher in A2 (481.9±49.2 nmol CHP/g ww) than in control worms (Ac=337.9±25.0 nmol CHP/g ww). Oxygen consumption was higher in worms acutely exposed to the lower copper concentration tested (A1=0.27±0.04 mg O2/g ww/h) than in the higher concentration (A2=0.14±0.01 mg O2/g ww/h). Changes in the swimming behavior of copper-exposed animals in both assays and edemas in the body wall of worms acutely exposed to copper were also observed. Results suggest that copper exposure favors reactive oxygen species generation and that enzymatic defense system is induced under chronic exposure, preventing oxygen consumption changes and lipid peroxidation and metahemoglobin formation. However, in acutely exposed worms, in spite of a transient peak of GST activity, no induction of antioxidant enzymes occurs, leading to morphological and physiological changes.

Antioxidant responses in different body regions of the polychaeta Laeonereis acuta (Nereididae) exposed to copper

Antioxidant enzymes, total antioxidant capacity (TOSC) and concentration of reactive oxygen species (ROS) were measured in anterior (A), middle (M) and posterior (P) body regions of Laeonereis acuta after copper (Cu; 62.5 microg/l) exposure. A catalase (CAT) activity gradient observed in control group (lowest in A, highest in P) was not observed in Cu exposed group. Glutathione-S-transferase (GST) activity in A region of Cu group was higher than in A region of the control group. DNA damage (comet assay) was augmented in the A region of Cu group. Since copper accumulation was similar in the different body regions, sensitivity to copper in A regions seems to be related to lowest CAT activity. In sum, copper exposure lowered TOSC, a result that at least in part can be related to lowering of antioxidant enzymes like CAT. DNA damage was induced in the anterior region, where a lower CAT activity was observed.

Current and Future Perspectives Using Biomarkers to Assess Pollution in Aquatic Ecosystems

The possibility of detecting aquatic pollution problems to take corrective decisions is intimately related to the organization levels of the living matter. Measurements at the biochemical or physiological levels detect more quickly and specifically the presence of several toxic compounds, allowing its utilization in a prospective way, until that deleterious effect reaches higher organization levels. The detection of environmental “hot spots” needs general nonspecific biomarkers if no previous information about the presence of specific pollutants exist. Since several xenobiotics can modify directly or indirectly the balance between pro-oxidant and antioxidant concentration, the determination of oxidative stress (DNA damage, protein oxidation, lipid peroxidation) in aquatic species is commonly used as a nonspecific biomarker. On the other hand, acetylcholinesterase activity or determination of metallothionein concentration (or expression) represents specific biomarkers that indicate the presence of anticholinesterasic compounds (organophosphorus pesticides or neurotoxins, for instance) and metals, respectively. The fact that animal populations inhabiting polluted areas can express responses qualitatively and quantitatively different from those of reference areas has lead to the “pollution induced community tolerance” approach that seems to be a valuable tool to compare toxicological responses of different aquatic populations.

Antioxidant Defenses and DNA Damage Induced by UV-A and UV-B Radiation in the Crab Chasmagnathus granulata (Decapoda, Brachyura)¶

Abstract The photoprotector role of pigment dispersion in the melanophores of the crab, Chasmagnathus granulata, against DNA and oxidative damages caused by UV-A and UV-B was investigated. Intact and eyestalkless crabs were used. In eyestalkless crabs, the dorsal epidermis of the cephalothorax (dispersed melanophores) and the epidermis of pereiopods (aggregated melanophores) were analyzed. Intact crabs showed only dispersed melanophores in the two epidermis. Antioxidant enzymes activity and lipoperoxidation content were analyzed after UV-A (2.5 J/cm2) or UV-B (8.6 J/cm2) irradiation. DNA damage was analyzed by single cell electrophoresis (comet) assay, after exposure to UV-B (8.6 J/cm2). UV-A radiation increased the glutatione-S-transferase activity in the pereiopods epidermis of eyestalkless crabs (P < 0.05). UV-B radiation induced DNA damage in the dorsal epidermis of eyestalkless crabs (P < 0.05). In pereiopod epidermis of eyestalkless crabs, there was no significant difference between control and UV-B–exposed crabs. In the pereiopods epidermis of eyestalkless, the control group showed higher scores of DNA damage and ∼50% of cellular viability. Because in eyestalkless and irradiated crabs the cellular viability was ∼5%, it was not possible to observe nuclei for determination of DNA damage. The findings show that melanophores can play a role in the defense against harmful effects of a momentary exposure to UV radiation.

Antioxidant mechanisms of the Nereidid Laeonereis acuta (Anelida: Polychaeta) to cope with environmental hydrogen peroxide.

Hydrogen peroxide (H2O2) is a naturally occurring prooxidant molecule, and its effects in the macroinvertebrate infauna were previously observed. The existence of a gradient of antioxidant enzymes activity (catalase [CAT], glutathione peroxidase [GPx], superoxide dismutase [SOD], and glutathione‐S‐transferase [GST]) and/or oxidative damage along the body of the estuarine polychaeta Laeonereis acuta (Polychaeta, Nereididae) was analyzed after exposure to H2O2. Because this species secretes conspicuous amounts of mucus, its capability in degrading H2O2 was studied. The results suggest that L. acuta deal with the generation of oxidative stress with different strategies along the body. In the posterior region, higher CAT and SOD activities ensure the degradation of inductors of lipid peroxidation such as H2O2 and superoxide anion (O2•−). The higher GST activity in anterior region aids to conjugate lipid peroxides products. In the middle region, the lack of high CAT, SOD, or GST activities correlates with the higher lipid hydroperoxide levels found after H2O2 exposure. Ten days of exposure to H2O2 also induced oxidative stress (lipid peroxidation and DNA damage) in the whole animal paralleled by a lack of CAT induction. The mucus production contributes substantially to H2O2 degradation, suggesting that bacteria that grow in this secretion provide this capability.

Sensitivity to microcystins: a comparative study in human cell lines with and without multidrug resistance phenotype.

Multidrug resistance (MDR) is an obstacle in cancer treatment. An understanding of how tumoral cells react to oxidants can help us elucidate the cellular mechanism involved in resistance. Microcystins are cyanobacteria hepatotoxins known to generate oxidative stress. The aim of this study was to compare the sensitivity to microcystins of human tumoral cell lines with (Lucena) and without (K562) MDR phenotype. Endpoints analyzed were effective microcystins concentration to 50% of exposed cells (EC50), antioxidant enzyme activity, lipid peroxidation, DNA damage, reactive oxygen species (ROS) concentration, and tubulin content. Lucena were more resistant and showed lower DNA damage than K562 cells (P < 0.05). Although microcystins did not alter catalase activity, a higher mean value was observed in Lucena than in K562 cells. Lucena cells also showed lower ROS concentration and higher tubulin content. The higher metabolism associated with the MDR phenotype should increase ROS concentration and make for an improved antioxidant defense against the toxic effects of microcystins.

Microcystin-induced oxidative stress in Laeonereis acuta (Polychaeta, Nereididae)

Oxidative stress induced by microcystins was evaluated in an estuarine worm, Laeonereis acuta (Nereididae). Ten organisms were exposed to lyophilized cells of a toxic Microcystisaeruginosa strain RST9501 ( approximately 2 microg/mL microcystins, MC); 10 were exposed to lyophilized cells of a nontoxic Aphanotece sp. strain RSMan92 and 10 were maintained without cyanobacterial cells. Exposure time was 48 h. The enzymatic antioxidant defenses, as well as the oxidative damage, were analyzed. Toxic and nontoxic cyanobacteria lowered catalase activity with no changes in glutathione reductase and glutathione-S-transferase activities. This may have led to toxin intracellular accumulation, which should favor oxidative stress generation, observed by the high lipid peroxide and DNA-protein crosslink levels in the group exposed to MC.

Annelids and Nematodes as Sentinels of Environmental Pollution

Worms like polychaetes, oligochaetes, and nematodes, have been widely used in studies of aquatic and soil pollution. Several ecotoxicological characteristics make these animals suitable for use as a biomonitor, including the fact that these organisms live in close contact with sediments. Also important is that they posses little mobility, which could be useful in biomonitoring programs since it can reflect local environmental problems. Several toxicological aspects are reviewed in these zoological groups, including their antioxidant responses and detoxifying mechanisms of heavy metals. Interestingly, the nematodes and oligochaetes species show induction of proteins different from the well-known metallothionein, including cysteine-rich proteins, and phytochelatins, this last group until recently believed to be present only in plants and fungi.

Photodynamic action of benzo[a]pyrene in K562 cells.

Benzo[a]pyrene (BaP) is ubiquitously distributed in the environment, being considered the most phototoxic element among polycyclic aromatic hydrocarbon (PAHs). In presence of oxygen, PAHs can act as a photosensitizer by means of promoting photo‐oxidation of biological molecules (photodynamic action, PDA). Thus, the present study analyzed the photodynamic action of BaP under UVA irradiation (BaP + UVA) and its oxidative effects on K562 cells. The evaluation of BaP kinetics showed that the highest intracellular concentration occurred after 18 h of incubation. Cell viability, reactive oxygen species (ROS) generation, lipid peroxidation, DNA damage (breaks and DNA–protein cross‐link [DNAPC]) were assessed after exposure to BaP, UVA and BaP plus UVA irradiation (BaP + UVA). Cell viability was decreased just after exposure to BaP + UVA. Lipid peroxidation and DNA breaks increased after BaP + UVA exposure, while the DNAPC increased after BaP, UVA and BaP + UVA exposure, suggesting that BaP + UVA effects were a consequence of both type II (producing mainly singlet oxygen) and type I (producing others ROS) mechanisms of PDA.