[Show abstract][Hide abstract] ABSTRACT: Fish are commonly exposed to environmental pollutants, which in turns could induce an oxidative stress. So, it is important to understand the effects and the responses elicited by these toxicants in fish species, being fish cell lines important tools for this purpose. Thus, the aim of the present study was to compare the effects of copper and UV-B radiation exposure on zebrafish hepatocytes (ZFL lineage) in terms of reactive oxygen species (ROS) levels, sulfhydril groups content and mRNA levels of important genes related to cellular response to toxic agents. Exposure of ZFL cells to UV-B radiation (23.3 mJ/cm(2)) significantly increased levels of intracellular ROS and mRNA of both superoxide dismutase isoforms (sod1 and sod2), three glutathione S-transferase isoforms (gstα, gstµ and gstπ) and a heat shock protein (hsp70). However, no changes in nonprotein sulfhydryl groups (NP-SH) content, as well as in the mRNA levels of genes related to glutathione (GSH) synthesis and recycling, were observed. Contrary to this, copper exposure (20 mg/L) diminished NP-SH content and increased the levels of mRNA of genes related to GSH synthesis (gclc and gs). Moreover, copper exposure increases the mRNA levels of some genes related to antioxidant defenses (gpx and gstπ), biotransformation reactions (cyp1a1) and protein repair (hsp70). In conclusion, these results demonstrated that both toxicants could increase ROS levels in ZFL cell line, but the responses are different, which could be related to activation of different signaling pathways.
Fish Physiology and Biochemistry 08/2014; · 1.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although the herbicide glyphosate [N-(phosphonomethyl)glycine] is not classified as an acethylcholinesterase inhibitor, some studies have reported reduction in the acethylcolinesterase activity after in vivo exposure to both its pure form and its commercial formulations. Considering this controversy, the objective of the present study was to investigate, in vitro, the effects of glyphosate exposure on cholinesterase activity of the brown mussel Perna perna and of two fish species: zebrafish Danio rerio and onesided livebearer Jenynsia multidentata. For this purpose, samples of different tissues (brain and muscle for fish; gills and muscle for mussel) were homogenized and pre-incubated with different glyphosate concentrations before cholinesterase activity determination. Results demonstrated that cholinesterase from different fractions of all species tested was inhibited by glyphosate. The concentrations of glyphosate that inhibits 50% of cholinesterase activity (IC(50)) ranged from 0.62mM for P. perna muscle to 8.43mM for J. multidentata brain. According to this, cholinesterase from mussel seems to be more sensitive to glyphosate exposure than those from the fish D. rerio and J. multidentata.
[Show abstract][Hide abstract] ABSTRACT: Marine ecosystems are typically subjected to a variety of stressors containing complex xenobiotics mixtures. This study aims to evaluate the responses of molecular and biochemical biomarkers in the mussel Mytilus edulis providing data for environmental monitoring programs development in Southern Brazil. Mussels were collected at a polluted site, near Patos Lagoon outfall, and at a control site. Gills, muscle and mantle samples were used for biomarker determinations. Mussels collected at the polluted site significantly increased superoxide dismutase (SOD) and glutathione S-transferase (GST) activities and decreased catalase (CAT) activity. Moreover, an increase in sod1, gstπ and hsp70 mRNA expression was observed. Overall, biochemical and molecular biomarkers responses were observed, but these responses varied depending on the analyzed tissue. These results indicate possible contaminants effects on organisms and the need for effective environmental monitoring programs in this ecosystem.
[Show abstract][Hide abstract] ABSTRACT: Neuropeptide Y (NPY) is one of the most potent stimulants of food intake in vertebrates, mammals and fish. However, the present
knowledge about feeding behaviour in fish is still limited and based on studies in a few species. The Brazilian flounder Paralichthys orbignyanus is being considered for aquaculture, and it is important to understand the mechanisms regulating feeding in order to improve
its performance in captivity. The objectives of this study were to clone NPY cDNA, evaluate the mRNA levels in different tissues
of flounder, and also evaluate brain NPY expression to associate food intake with NPY expression levels. A 597 bp NPY cDNA
was cloned from Brazilian flounder brain. NPY expression was detected in all the peripheral tissues analysed. No significant
differences were observed in brain NPY gene expression over 24 h after food intake at a temperature of 15 ± 3°C. No correlation
was observed among plasma glucose, total protein, cholesterol, triglycerides and NPY expression levels during this 24 h period.
On the other hand, mRNA levels were increased after two weeks of fasting at elevated temperatures. Our results suggest that
NPY mRNA levels in Brazilian flounder are affected by temperature.
KeywordsFood intake-gene expression-NPY-
Journal of Biosciences 09/2010; 35(3):405-413. · 1.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Neuropeptide Y (NPY) is one of the most potent stimulants of food intake in vertebrates, mammals and fish. However, the present knowledge about feeding behaviour in fish is still limited and based on studies in a few species. The Brazilian flounder Paralichthys orbignyanus is being considered for aquaculture, and it is important to understand the mechanisms regulating feeding in order to improve its performance in captivity. The objectives of this study were to clone NPY cDNA, evaluate the mRNA levels in different tissues of flounder, and also evaluate brain NPY expression to associate food intake with NPY expression levels. A 597 bp NPY cDNA was cloned from Brazilian flounder brain. NPY expression was detected in all the peripheral tissues analysed. No significant differences were observed in brain NPY gene expression over 24 h after food intake at a temperature of 15 +or- 3 degrees C. No correlation was observed among plasma glucose, total protein, cholesterol, triglycerides and NPY expression levels during this 24 h period. On the other hand, mRNA levels were increased after two weeks of fasting at elevated temperatures. Our results suggest that NPY mRNA levels in Brazilian flounder are affected by temperature.
Journal of Biosciences 09/2010; 35(3):405-13. · 1.76 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The K562 cell line (chronic myeloid leukemia), sensitive to chemotherapy (non-MDR), and the Lucena cell line, resistant to chemotherapy (MDR) were investigated. The results suggest that both cell lines possess CD34+CD38- profiles of hematopoietic stem cell markers. The promoter regions of ABCB1, ABCG2 and ABCC1 genes contain binding sites for the Oct-4 transcripton factor, which is also considered a marker of tumor stem cells. Lucena cells showed an over-expression of the ABCB1 gene and a high expression of the Oct-4, ABCG2 and ABCC1 genes as compared to K562 cells.
Leukemia research 12/2009; 34(6):757-62. · 2.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Copper is an essential metal to aquatic animals, but it can be toxic when in elevated concentrations in water. The objective of the present study was to analyze copper effects in zebrafish hepatocytes (ZFL cell-line). The number of viable cells and copper accumulation were determined in hepatocytes exposed in vitro to different copper concentrations (5-30mgCu/L). Intracellular reactive oxygen species (ROS) formation, total antioxidant capacity against peroxyl radicals, and expression of genes related do DNA repair system were also measured in hepatocytes exposed to 5 and 20mgCu/L. After 24h of exposure, hepatocytes showed an exponential kinetics of copper accumulation. Copper exposure (24 and 48h) significantly reduced hepatocyte number in all concentrations tested, except at the lowest one (5mgCu/L). Exposure to 20mgCu/L for 6, 12 and 24h significantly increased intracellular ROS formation. However, no significant change in total antioxidant capacity was observed. After 12 and 24h of exposure to 20mgCu/L, a significant decrease in expression of p53 and CDKI genes was observed. Conversely, expression of Gadd45alpha, CyclinG1 and Bax genes was significantly induced after 24h of exposure to 20mgCu/L. In hepatocytes exposed to 5mgCu/L, any significant alteration in expression of these genes was observed. In a broad view, most of genes encoding for DNA repair proteins were inhibited after copper exposure, especially in hepatocytes exposed to 20mgCu/L. Taken all together, results obtained suggest that the increased intracellular ROS formation induced by copper exposure would be responsible for the alteration in gene expression pattern observed.
[Show abstract][Hide abstract] ABSTRACT: 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.
Ecotoxicology and Environmental Safety 09/2008; 72(2):388-93. · 2.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The objective of this study was to evaluate the time-course effects of UV-B exposure on expression of genes involved in the DNA repair system of zebrafish (Danio rerio) hepatocytes, a highly competent species in terms of damage repair induced by UV radiation. For gene expression analysis (RT-PCR), cells were exposed to 23.3 mJ cm(-2) UV-B, which was the dose that affected viable cell number (reduction of 30% when compared with the control group) and produced no visual alteration on cell morphology. The early response observed (6 h) showed induction in the expression of the CDKI gene (cyclin-dependent kinase inhibitor) and genes related to DNA damage repair (mainly XPC and DDB2), while the late response observed (24 h) was more related to up-regulation of p53 and genes involved in cell cycle arrest (gadd45a, cyclinG1). In all times analyzed, the anti-apoptotic gene Bcl-2 was down-regulated. Another interesting result observed was the up-regulation of the Apex-1 gene after UV-B exposure, which could indicate the induction of oxidative lesions in the DNA molecule. In conclusion, these results demonstrate an activation of the DNA repair system in hepatocytes of zebrafish exposed to UV-B radiation, mainly involving the participation of p53.
Photochemistry and Photobiology 09/2008; 85(1):220-6. · 2.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The objective of this study was to evaluate the effects of the exposure to cadmium on the antioxidant responses in the polychaeta Laeonereis acuta. The worms were submitted to 0, 5, and 100 microg of Cd/L during a period of test of 7 days. Cadmium was significantly (p<0.05) accumulated in L. acuta in both concentrations assayed, but the concentration of reactive oxygen and nitrogen species (RONS) increased (p<0.05) only in the group submitted to the highest concentration of cadmium (100 microg/L). At this concentration, a decrease in the activity of the superoxide dismutase and an increase of glutathione-S-transferase activity (p<0.05) was observed. The levels of both lipid peroxides and the activities of catalase and glutathione peroxidase were not affected (p>0.05) by the exposition to cadmium. Thus, cadmium can augment RONS levels and can interfere with the antioxidant defense system of the polychaete L. acuta, although cadmium does not directly induce oxidative stress unlike copper and iron.
Ecotoxicology and Environmental Safety 05/2008; 70(1):115-20. · 2.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Environment International 06/2007; 33(4):559-64. · 5.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Microcystins are usually the predominant cyanotoxins present in both drinking and recreational waters after cyanobacterial blooms. Their classic toxic effect is hepatotoxicity through inhibition of serine/threonine phosphatases. However, recent studies also reported oxidative stress generation and disruption of ion regulation in aquatic organisms after microcystins exposure. In the present study, aqueous extracts of Microcystis aeruginosa were administered to the estuarine crab Chasmagnathus granulatus (Decapoda, Brachyura) by gavage in variable doses (from 34 to 860 microg kg(-1)) and exposure times (6, 12, and 72 h). A control group was exposed to saline solution. Analyzed variables included oxygen consumption, lipid peroxidation (LPO), enzyme activities (glutathione S-transferases or GST; alanine aminotransferase or ALT; aspartate aminotransferase or AST; and lactate dehydrogenase or LDH), glycogen, and microcystins content. Oxygen consumption increased in organisms exposed for 12h to 860 microg kg(-1) of microcystins and a similar result was observed after 72 h at doses equal to or higher than 34 microg kg(-1). LPO levels increased in doses equal to or higher than 34 microg kg(-1) after 72 h. GST and LDH activities increased after 12 h (at a dose of 860 microg kg(-1)), but ALT and AST activities remained unaltered in all experimental conditions. Glycogen content decreased after 72 h exposure at doses equal to or higher than 172 microg kg(-1). After 12h of exposure to 860 microg kg(-1) of microcystins, the concentration found in the hepatopancreas of C. granulatus was 13.17+/-0.56 microg kg(-1). In crabs exposed to doses higher than 172 microg kg(-1) during 72 h this value raised to 32.14+/-4.12 microg kg(-1). The obtained results indicated that microcystins exposure led the tissue to an oxidative stress condition (high LPO levels), at least in part favored by the augment of oxygen consumption, altering the glycogen metabolism. GST responses were only observed in the short-term experiment (12 h) and no effect on classical markers of vertebrate liver damage (ALT and AST) was observed. Although the hepatopancreas from C. granulatus accumulated a relatively low concentration of toxins, it was enough to induce physiological and biochemical disturbances.
Ecotoxicology and Environmental Safety 11/2006; 65(2):201-8. · 2.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The enzyme glutamate cysteine ligase (GCL) is the rate-limiting enzyme in glutathione (GSH) synthesis and is formed by a catalytic (GCLC) and a modulatory subunit (GCLM). Some studies have demonstrated that environmental pollutants can regulate the expression of these subunits. Despite the importance of these genes in toxicological responses, no sequences are available for the GCL subunits in annelids. The present study reports, for the first time, the cDNA sequence for the GCLC in an annelid species, the polychaete Laeonereis acuta (Nereididae). The deduced amino acid sequence of L. acuta GCLC showed homology with other animal species, and was used to infer a phylogenetic tree with GCLC amino acid sequences from other taxonomic groups. Exposure to cadmium (100 and 1000 microg Cd/L) during 14 days augmented the level of L. acuta GCLC transcripts in a dose-dependent manner. These gene expression results can be related to the known cadmium effect on GSH depletion. Since a number of contaminants can also exert their toxicity through this mechanism, GCLC gene expression might be applied not only for cadmium biomonitoring, but also for a wide range of contaminants that directly or indirectly promote the same effect in the cellular GSH content.
Comparative Biochemistry and Physiology Part C Toxicology & Pharmacology 09/2006; 143(4):410-5. · 2.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Some effects of cadmium exposure (100 microg/L for 4, 8, 12, and 24 h) on the estuarine polychaete Laeonereis acuta (Nereididae) were evaluated. This polychaete was able to accumulate cadmium in the body, with the metal stored mainly in the cytosolic fraction (>10 kDa). Activity of the antioxidant enzymes superoxide dismutase, glutathione S-transferase, and glutathione reductase (GR) as well as the total oxyradical scavenger capacity, the glutamate cysteine ligase catalytic subunit gene expression, and the metallothionein-like proteins content were not affected by cadmium at any exposure time tested. Catalase (CAT) activity, however, was significantly lower (p < 0.05) in worms treated with cadmium compared with that in controls after 8 h of exposure. At the same exposure time, lipid peroxide levels were increased (p < 0.05) in worms exposed to cadmium compared with those in control worms. Interestingly, CAT and GR activities decreased over time (p < 0.05) independent of cadmium treatment, which is a result that could be attributed to starvation. The effects caused by cadmium in the present study were observed only after 8 h of exposure, demonstrating that cadmium can generate oxidative stress.
Environmental Toxicology and Chemistry 06/2006; 25(5):1337-44. · 2.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Comparative Biochemistry and Physiology Part C Toxicology & Pharmacology 03/2006; 142(3-4):293-300. · 2.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.