In vitro exposure to fullerene C(60) influences redox state and lipid peroxidation in brain and gills from Cyprinus carpio (Cyprinidae).

Studies concerning the impact of nanomaterials, especially fullerene (C(60) ), in fresh water environments and their effects on the physiology of aquatic organisms are still scarce and conflicting. We aimed to assess in vitro effects of fullerene in brain and gill homogenates of carp Cyprinus carpio, evaluating redox parameters. A fullerene suspension was prepared by continued stirring under fluorescent light during two months. The suspension concentration was measured by total carbon content and ultraviolet-visible spectroscopy nephelometry. Characterization of C(60) aggregates was performed with an enhanced dark-field microscopy system and transmission electronic microscopy. Organ homogenates were exposed during 1, 2, and 4 h under fluorescent light. Redox parameters evaluated were reduced glutathione and oxidized glutathione, cysteine and cystine, total antioxidant capacity; activity of the antioxidant enzymes glutathione S-transferase and glutathione reductase (GR), and lipid peroxidation (TBARS assay). Fullerene induced a significant increase (p < 0.05) in lipid peroxidation after 2 h in both organs and reduced GR activity after 1 h (gills) and 4 h (brain) and antioxidant capacity after 4 h (brain). Levels of oxidized glutathione increased in the brain at 1 h and decreased at 2 h as well. Given these results, it can be concluded that C(60) can induce redox disruption via thiol/disulfide pathway, leading to oxidative damage (higher TBARS values) and loss of antioxidant competence.

Induction of phase II enzymes and hsp70 genes by copper sulfate through the electrophile-responsive element (EpRE): insights obtained from a transgenic zebrafish model carrying an orthologous EpRE sequence of mammalian origin.

We have evaluated the homology of the electrophile-responsive element (EpRE) core sequence, a binding site for the Nrf2 transcription factor, in the proximal promoters of the mouse and zebrafish glutathione-S-transferase (gst), glutamate cysteine ligase catalytic subunit (gclc) and heat shock protein 70 (hsp70) genes. The EpRE sites identified for both species in the three analyzed genes showed a high similarity with the putative EpRE core sequence. We also produced a transgenic zebrafish model carrying a transgene comprised of the luciferase (luc) reporter gene under transcriptional control of a mouse EpRE sequence. This transgenic model was exposed to copper sulfate, and the reporter gene was significantly activated. The endogenous gst, gclc and hsp70 zebrafish genes were analyzed in the EpRE-Luc transgenic zebrafish and showed an expression pattern similar to that of the reporter transgene used. Our results demonstrate that EpRE is conserved between mouse and zebrafish for detoxification-related genes and that the development of genetically modified models using this responsive element to drive the expression of reporter genes can be an important tool in understanding the action mechanism of aquatic pollutants.

Effects of arsenic (As) exposure on the antioxidant status of gills of the zebrafish Danio rerio (Cyprinidae).

In fishes, arsenic (As) is absorbed via the gills and is capable of causing disturbance to the antioxidant system. The objective of present study was to evaluate antioxidant responses after As exposure in gills of zebrafish (Danio rerio, Cyprinidae). Fish were exposed for 48 h to three concentration of As, including the highest As concentration allowed by current Brazilian legislation (10 microg As/L). A control group was exposed to tap water (pH 8.0; 26 degrees C; 7.20 mg O(2)/L). As exposure resulted in (1) an increase (p<0.05) of glutathione (GSH) levels after exposure to 10 and 100 microg As/L, (2) an increase of the glutamate cysteine ligase (GCL) activity in the same concentrations (p<0.05), (3) no significant differences in terms of glutathione reductase, glutathione-S-transferase and catalase activities; (4) a significantly lower (p<0.05) oxygen consumption after exposure to 100 microg As/L; (4) no differences in terms of oxygen reactive species generation and lipid peroxidation content (p>0,05). In the gills, only inorganic As was detected. Overall, it can be concluded that As affected the antioxidant responses increasing GCL activity and GSH levels, even at concentration considered safe by Brazilian legislation.

Initial results in the development of a reporter cell line for toxicology studies at gene expression level: activation of the electrophile-responsive element by copper and methyl parathion.

Induction of many genes encoding detoxifying enzymes and antioxidant proteins is mediated through a common mechanism, which is controlled by electrophile-responsive elements (EpRE) within the regulatory region of those genes. Copper and methyl parathion are environmental pollutants known to induce the expression of EpRE-mediated genes. In order to evaluate the molecular response triggered by these pollutants, a stable cell line was produced, which carries a transgene comprised of the green fluorescent protein (GFP) reporter gene under transcriptional control of the mouse glutathione-S-transferase (gst1) electrophile-responsive element fused to the mouse metallothionein (mt1) minimal promoter. The rat HTC hepatoma cells were transfected with the EpREmt-GFP construct and successfully selected with G418 antibiotic. EpREmt-GFP HTC cells were treated with 0.002 mg L(-1), 0.02 mg L(-1), 0.2 mg L(-1) and 2 mg L(-1) copper sulfate and 0.001 mg L(-1), 0.01 mg L(-1), 0.1 mg L(-1) and 1 mg L(-1) methyl parathion for 48 h. GFP expression was directly quantified in living cells using a microplate fluorimeter. GFP expression was significantly increased in higher concentrations of both pollutants, showing a 1.80- and 2.58-fold induction of GFP at 2mg copper L(-1) and 1mg methyl parathion L(-1), respectively (p<0.01). The results obtained in the present study demonstrate that the EpREmt-GFP HTC cell line can be an interesting model for further development for the study of the cellular response to aquatic pollutants as well as a new tool for environmental monitoring at the molecular level.

Antioxidant responses and reactive oxygen species generation in different body regions of the estuarine polychaeta Laeonereis acuta (Nereididae).

The aim of this study was to analyze the total antioxidant capacity (TOSC), generation of reactive oxygen species (ROS) and lipid peroxidation (LPO) in the different body regions of the estuarine polychaeta Laeonereis acuta (Nereididae) sampled at non-polluted (NOPOL) and polluted (POL) sites from Lagoa dos Patos (Southern Brazil). Organisms collected at POL during summer showed similar (p>0.05) TOSC values along the body, but worms collected at NOPOL presented higher (p<0.05) TOSC values in the posterior (P) region in respect of anterior (A) region and middle (M) region. TOSC in the P region at NOPOL was higher (p<0.05) compared with the same body region of worms at POL. In summer, ROS concentration was higher in A and M regions of worms at POL in respect of the organisms at NOPOL. During winter all the regions showed higher ROS in worms sampled at POL. It was registered absence of season influence on LPO content, but in the P region at NOPOL in summer there were lower LPO levels compared with the others regions (p<0.05). In vitro assays showed that P region, despite a higher basal ROS, presented a higher competence to cope with pro-oxidants compared with A and M regions (p<0.05), corroborating the field results. A lower proteic sulfhydril content was observed in P in respect of the other regions (p<0.05) supporting the idea of a highest oxidant condition in this region. The results indicate that worms collected at the POL site are confronted to higher ROS concentrations, affecting its antioxidant capacity, a result that depends of body regions.

Toxicological responses in Laeonereis acuta (annelida, polychaeta) after arsenic exposure.

Several environmental pollutants, including metals, can induce oxidative stress. So, the objective of this study was to evaluate the effects of arsenic (As(III), as As(2)O(3)) on the antioxidant responses in the polychaete Laeonereis acuta. Worms were exposed to two environmentally relevant concentrations of As, including the highest previously allowed by Brazilian legislation (50 microg As/l). A control group was kept in saline water (10 per thousand) without added metal. It was observed that: (1) a peak concentration of lipid peroxide was registered after 2 days of exposure to 50 microg As/l (61+/-3.2 nmol CHP/g wet weight) compared to the control group (43+/-4.5 nmol CHP/g wet weight), together with a lowering of the activity of the antioxidant enzyme catalase (-47 and -48%, at 50 or 500 microg As/l respectively) and a higher superoxide dismutase activity (+305% at 50 microg As/l with respect to the control group); (2) a lower conjugation capacity through glutathione-S-transferase activity was observed after 7 days of exposure to 50 microg As/l (-48% compared to the control group); (3) a significant increase in As concentration was verified after 1 week of exposure to both As concentrations (50 and 500 microg/l); (4) worms exposed to As showed a limited accumulation of related methylated As species and the levels of non-toxic As species like arsenobetaine (AsB) and arsenocholine (AsC) remained unchanged during the exposure period when compared with the controls. Overall, it can be concluded that As interfered in the antioxidant defense system of L. acuta, even at low concentrations (50 microg/l) that Brazilian legislation previously considered safe. The fact that worms exposed to As showed high levels of methylated As species indicates the methylation capability of L. acuta, although the high levels of inorganic As suggest that not all the administered As(III) (as As(2)O(3)) is completely removed or biotransformed after 7 days of exposure.

Pollution biomarkers in estuarine animals: critical review and new perspectives.

In this review, recent developments in monitoring toxicological responses in estuarine animals are analyzed, considering the biomarker responses to different classes of pollutants. The estuarine environment imposes stressful conditions to the organisms that inhabit it, and this situation can alter their sensitivity to many pollutants. The specificity of some biomarkers like metallothionein tissue concentration is discussed in virtue of its dependence on salinity, which is highly variable in estuaries. Examples of cholinesterase activity measurements are also provided and criteria to select sensitive enzymes to detect pesticides and toxins are discussed. Regarding non-specific biomarkers, toxic responses in terms of antioxidant defenses and/or oxidative damage are also considered in this review, focusing on invertebrate species. In addition, the presence of an antioxidant gradient along the body of the estuarine polychaete Laeonereis acuta (Nereididae) and its relationship to different strategies, which deal with the generation of oxidative stress, is reviewed. Also, unusual antioxidant defenses against environmental pro-oxidants are discussed, including the mucus secreted by L. acuta. Disruption of osmoregulation by pollutants is of paramount importance in several estuarine species. In some cases such as in the estuarine crab Chasmagnathus granulatus, there is a trade off between bioavailability of toxicants (e.g. metals) and their interaction with key enzymes such as Na(+)-K(+)-ATPase and carbonic anhydrase. Thus, the metal effect on osmoregulation is also discussed in the present review. Finally, field case studies with fish species like the croaker Micropogonias furnieri (Scianidae) are used to illustrate the application of DNA damage and immunosuppressive responses as potential biomarkers of complex mixture of pollutants.

Application of public-domain statistical analysis software for evaluation and comparison of comet assay data.

A novel approach for statistical analysis of comet assay data (i.e.: tail moment) is proposed, employing public-domain statistical software, the R system. The analytical strategy takes into account that the distribution of comet assay data, like the tail moment, is usually skewed and do not follow a normal distribution. Probability distributions used to model comet assay data included: the Weibull, the exponential, the logistic, the normal, the log normal and log-logistic distribution. In this approach it was also considered that heterogeneity observed among experimental units is a random feature of the comet assay data. This statistical model can be characterized with a location parameter m(ij), a scale parameter r and a between experimental units variability parameter theta. In the logarithmic scale, the parameter m(ij) depends additively on treatment and random effects, as follows: log(m(ij)) = a0 + a1x(ij) + b(i), where exp(a0) represents approximately the mean value of the control group, exp(a1) can be interpreted as the relative risk of damage with respect to the control group, x(ij) is an indicator of experimental group and exp(b(i)) is the individual risk effects assume to follows a Gamma distribution with mean 1 and variance theta. Model selection is based on Akaike's information criteria (AIC). Real data coming from comet analysis of blood samples taken from the flounder Paralichtys orbignyanus (Teleostei: Paralichtyidae) and from samples of cells suspension obtained from the estuarine polychaeta Laeonereis acuta (Nereididae) were employed. This statistical approach showed that the comet assay data should be analyzed under a modeling framework that take into account the important features of these measurements. Model selection and heterogeneity between experimental units play central points in the analysis of these data.

Antioxidant properties of the mucus secreted by Laeonereis acuta (Polychaeta, Nereididae): a defense against environmental pro-oxidants?

Polychaeta species like Laeonereis acuta (Nereididae) usually secrete great amounts of mucus that wrap the animal inside. Taking into account that fungi action in the sediment and UV radiation acting on dissolved organic matter in the water produces reactive oxygen species (ROS) like hydrogen peroxide (H(2)O(2)), it was considered that the mucus secretion could represent an antioxidant defense against environmental ROS. Antioxidant enzymes (catalase-CAT; superoxide dismutase-SOD; glutathione peroxidase-GPx and glutathione-S-transferase-GST) and total antioxidant capacity (TOSC) were determined in worms and mucus secretion. Higher (p<0.05) CAT, GPx and TOSC values were registered in mucus samples respect worms, SOD activity was similar (p>0.05) in both kind of samples, and absence of GST activity was observed in mucus samples, suggesting absence of catalyzed phase II reactions. In assays conducted with hepatoma cell lines exposed to H(2)O(2), it was verified that: (1) mucus co-exposure significantly (p<0.05) lowered DNA damage induced by H(2)O(2); (2) ROS production was significantly (p<0.05) reduced when cells were exposed simultaneously with mucus samples and H(2)O(2) respect H(2)O(2) alone. It can be concluded that the mucus production contributes substantially to the antioxidant defense system of the worm against environmental ROS through the interception or degradation of H(2)O(2), peroxyl and hydroxyl radicals.

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

Hydrogen peroxide (H(2)O(2)) 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 H(2)O(2). Because this species secretes conspicuous amounts of mucus, its capability in degrading H(2)O(2) 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 H(2)O(2) and superoxide anion (O(2)(.-)). 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 H(2)O(2) exposure. Ten days of exposure to H(2)O(2) 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 H(2)O(2) degradation, suggesting that bacteria that grow in this secretion provide this capability.

Antioxidant defenses and DNA damage induced by UV-A and UV-B radiation in the crab Chasmagnathus granulata (Decapoda, Brachyura).

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 approximately 50% of cellular viability. Because in eyestalkless and irradiated crabs the cellular viability was approximately 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.

Oxidative stress in Laeonereis acuta (Polychaeta, Nereididae): environmental and seasonal effects.

Laeonereis acuta was seasonally collected in an industrially polluted site (P) and in an unpolluted site (UP) at the Patos Lagoon estuary (southern Brazil). Glutathione S-transferase (GST), superoxide dismutase (SOD) and catalase (CAT) activity (U/mg protein) was determined in five groups of worms from each site. Metallothionein (MT - mol GSH/g ww) and lipid peroxides content (LPO - nmoles of cumene hydroperoxide/g ww) were also measured. Annual mean values for CAT (UP=3.7+/-0.3; P=5.7+/-0.6), GST (UP=0.034+/-0.003; P=0.045+/-0.004) and MT (UP=0.15+/-0.02; P=0.23+/-0.03) were higher (p<0.05) in worms from the P site. In autumn, CAT activity was higher (p<0.05) in worms from the P site (7.6 +/- 1.3) than in those from the UP site (3.6 +/- 0.4). In summer, MT concentration was higher in worms from the P site (0.37 +/- 0.03) than in those from the UP site (0.19 +/- 0.01). No significant difference (p>0.05) in the LPO content was observed in worms from the different sites or collected in different seasons. These results indicate that worms from the polluted site showed higher antioxidant responses than those from the unpolluted site, sufficient to prevent oxidative damage in terms of LPO.