Biomarker responses as indication of contaminant effects in Oreochromis niloticus

The current study investigated oxidative stress parameters (enzymes activities, metallothionein content and lipid peroxidation) in freshwater fish, Oreochromis niloticus, tilapia exposure to Monjolinho River (in 4 months of year: January, April, July and November). One critical site in Monjolinho River (site B) was assessed in comparison to a reference site (site A). Water pH and oxygen concentration was lower than that recommended by CONAMA (Brazilian National Environmental Committee), resolution 357/2005 for protection of aquatic communities, and ammonium and the metals Cu, Zn, Mn and Fe (on all months) concentrations were higher than the maximum concentration recommended. Glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities were significantly decreased in liver and muscle in tila. pia from Monjolinho River, throughout the year, in relation to reference except in gills that SOD activity increased. Glutathione S-transferase (GST) activity was significantly increased in liver of the tilapia from Monjolinho River in all sites, in relation to reference except in gills that GST activity increased in July and decreased in November, suggesting that GST activity could be induced to neutralize the pollutants toxicity. On the other hand, GST activity was significantly decreased in white muscle indicating a toxic effect of pollutants, resulting in a decreased ability of tilapia to perform defense reactions associated to GSTs. The decrease of catalase (CAT) activity in gills of the O. niloticus together with the increase of SOD activity, could explain the increased lipid peroxidation (LPO) level in this organ. Metallothionein levels in liver and gills were significantly high in all sites. Results indicate that the exposure to metals caused severe damage to tissues; despite the consensually assumed antioxidant induction as a sign of exposure to contaminants the effects seem in part to be mediated by suppression of antioxidant system with SOD, CAT and GPx as potential candidates for tissues toxicity biomarkers of pollutants. (c) 2012 Elsevier Ltd. All rights reserved.

Determination of relative in vivo osteoconductivity of modified potassium fluorrichterite glass-ceramics compared with 45S5 bioglass

Potassium fluorrichterite (KNaCaMg5Si8O22F2) glass-ceramics were modified by either increasing the concentration of calcium (GC5) or by the addition of P2O5 (GP2). Rods (2 x 4 mm) of stoichiometric fluorrichterite (GST), modified compositions (GC5 and GP2) and 45S5 bioglass, which was used as the reference material, were prepared using a conventional lost-wax technique. Osteoconductivity was investigated by implantation into healing defects in the midshaft of rabbit femora. Specimens were harvested at 4 and 12 weeks following implantation and tissue response was investigated using computed microtomography (mu CT) and histological analyses. The results showed greatest bone to implant contact in the 45S5 bioglass reference material at 4 and 12 weeks following implantation, however, GST, GC5 and GP2 all showed direct bone tissue contact with evidence of new bone formation and cell proliferation along the implant surface into the medullary space. There was no evidence of bone necrosis or fibrous tissue encapsulation around the test specimens. Of the modified potassium fluorrichterite compositions, GP2 showed the greatest promise as a bone substitute material due to its osteoconductive potential and superior mechanical properties.

Sewage sludge does not induce genotoxicity and carcinogenesis

Through a series of experiments, the genotoxic/mutagenic and carcinogenic potential of sewage sludge was assessed. Male Wistar rats were randomly assigned to four groups: Group 1 - negative control; Group 2 - liver carcinogenesis initiated by diethylnitrosamine (DEN; 200 mg/kg i.p.); Group 3 and G4-liver carcinogenesis initiated by DEN and fed 10,000 ppm or 50,000 ppm of sewage sludge. The animals were submitted to a 70% partial hepatectomy at the 3rd week. Livers were processed for routine histological analysis and immunohistochemistry, in order to detect glutathione S-transferase positive altered hepatocyte foci (GST-P+ AHF). Peripheral blood samples for the comet assay were obtained from the periorbital plexus immediately prior to sacrificing. Polychromatic erythrocytes (PCEs) were analyzed in femoral bone-marrow smears, and the frequencies of those micronucleated (MNPCEs) registered. There was no sewage-sludge-induced increase in frequency of either DNA damage in peripheral blood leucocytes, or MNPCEs in the femoral bone marrow. Also, there was no increase in the levels of DNA damage, in the frequency of MNPCEs, and in the development of GST-P AHF when compared with the respective control group.

Assessment of the sublethal toxicity of organochlorine pesticide endosulfan in juvenile common carp (Cyprinus carpio)

This study is aimed at evaluating the sublethal effects of endosulfan (EDS) in juvenile common carp (Cyprinus carpio). For this purpose, fish were exposed for 15 days to the technical EDS (95% pure) diluted in dimethyl sulfoxide (DMSO) 0.1% of the total volume in water solution in a semi-static system at sublethal concentration (1 mu g/L). Subsequently, the liver somatic index (LSI) and factor condition (K) were determined. The total cytocrome P450 (CYP), CYP1A isoform, and the ethoxyresorufin-O-deethylase (EROD) activity were determined from the hepatic microsomal fraction as well as the activity of the oxidative stress enzyme system such as superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), glutathione peroxidase (GP(X)), glutathione reductase (GR), and glucose-6-phosphate dehydrogenase (G6PDH). Among the parameters assessed, EDS at the sublethal concentration in subchronic exposure caused significant changes in liver somatic indices as well as induction of the phase I biotransformation system and oxidative stress in juvenile common carp (Cyprinus carpio). Thus, it is seen that the use of biochemical biomarkers of environmental contamination in this study proved to be an extremely important tool for detecting the adverse effects of xenobiotics in the aquatic environment, even at low concentration.

EVALUATION OF GLUTATHIONE S-TRANSFERASE GSTM1 AND GSTT1 POLYMORPHISMS AND METHYLMERCURY METABOLISM IN AN EXPOSED AMAZON POPULATION

Over the last decades, the presence of methylmercury (MeHg) in the Amazon region of Brazil and its adverse human health effects have given rise to much concern. The biotransformation of MeHg occurs mainly through glutathione (GSH) in the bile mediated by conjugation with glutathione S-transferases (GST). Epidemiological evidence has shown that genetic polymorphisms may affect the metabolism of MeHg. The aim of this study was to evaluate the association between GST polymorphisms, GSH, and Hg levels in blood (B-Hg) and in hair (H-Hg) of an Amazon population chronically exposed to the metal through fish consumption. Blood and hair samples were collected from 144 volunteers (71 men, 73 women). B-Hg and H-Hg levels were determined by inductively coupled plasma-mass spectrometry, and GSH levels were evaluated by a spectrophotometric method. GSTM1 and T1 genotyping evaluation were carried out by multiplex polymerase chain reaction (PCR). Mean levels of B-Hg and H-Hg were 37.7 +/- 24.5 mu g/L and 10.4 +/- 7.4 mu g/g, respectively; GSH concentrations ranged from 0.52 to 2.89 mu M/ml of total blood. Distributions for GSTM1/T1, GSTM1/GSTT1*0, GSTM1*0/T1, and GSTM1*0/GSTT1*0 genotypes were 35.4, 22.2, 25.0, and 17.4%, respectively. GSTT1 genotype carriers presented lower levels of B-Hg and H-Hg when compared to other genotypes carriers. In addition, GSTM1*0/GSTT1*0 individuals presented higher Hg levels in blood and hair than subjects presenting any other genotypes. There appeared to be no evidence of an effect of polymorphisms on GSH levels. Therefore, our data suggest that GST polymorphisms may be associated with MeHg detoxification.

ENVIRONMENTAL TOBACCO SMOKE INDUCES OXIDATIVE STRESS IN DISTINCT BRAIN REGIONS OF INFANT MICE

Environmental tobacco smoke (ETS) leads to the death of 600,000 nonsmokers annually and is associated with disturbances in antioxidant enzyme capacity in the adult rodent brain. However, little is known regarding the influence of ETS on brain development. The aim of this study was to determine levels of malonaldehyde (MDA) and 3-nitrotyrosine (3-NT), as well as enzymatic antioxidant activities of glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), and superoxide dismutase (SOD), in distinct brain structures. BALB/c mice were exposed to ETS twice daily for 1 h from postnatal day 5 through postnatal day 18. Acute exposure was performed for 1 h on postnatal day 18. Mice were euthanized either immediately (0) or 3 h after the last exposure. Immediately after an acute exposure there were higher GR and GST activities and MDA levels in the hippocampus, higher GPx and SOD activities in the prefrontal cortex, and higher GST activity and MDA levels in the striatum and cerebellum. Three hours later there was an increase in SOD activity and MDA levels in the hippocampus and a decrease in the activity of all enzymes in the prefrontal cortex. Immediately after final repeated exposure there were elevated levels of GST and GR activity and decreased GPx activity in the hippocampus. Moreover, a rise was found in GPx and GST activities in the prefrontal cortex and increased GST and GPx activity in the striatum and cerebellum, respectively. After 3 h the prefrontal cortex showed elevated GR and GST activities, and the striatum displayed enhanced GST activity. Data showed that enzymatic antioxidant system in the central nervous system responds to ETS differently in different regions of the brain and that a form of adaptation occurs after several days of exposure.

Coffee and Caffeine Protect against Liver Injury Induced by Thioacetamide in Male Wistar Rats

Coffee intake has been inversely related to the incidence of liver diseases, although there are controversies on whether these beneficial effects on human health are because of caffeine or other specific components in this popular beverage. Thus, this study evaluated the protective effects of coffee or caffeine intake on liver injury induced by repeated thioacetamide (TAA) administration in male Wistar rats. Rats were randomized into five groups: one untreated group (G1) and four groups (G2G5) treated with the hepatotoxicant TAA (200 similar to mg/kg b.w., i.p.) twice a week for 8 similar to weeks. Concomitantly, rats received tap water (G1 and G2), conventional coffee (G3), decaffeinated coffee (G4) or 0.1% caffeine (G5). After 8 similar to weeks of treatment, rats were killed and blood and liver samples were collected. Conventional and decaffeinated coffee and caffeine intake significantly reduced serum levels of alanine aminotransferase (ALT) (p similar to<similar to 0.001) and oxidized glutathione (p similar to<similar to 0.05), fibrosis/inflammation scores (p similar to<similar to 0.001), collagen volume fraction (p similar to<similar to 0.01) and transforming growth factor beta-1 (TGF-beta 1) protein expression (p similar to=similar to 0.001) in the liver from TAA-treated groups. In addition, conventional coffee and caffeine intake significantly reduced proliferating cellular nuclear antigen (PCNA) S-phase indexes (p similar to<similar to 0.001), but only conventional coffee reduced cleaved caspase-3 indexes (p similar to<similar to 0.001), active metalloproteinase 2 (p similar to=similar to 0.004) and the number of glutathione S-transferase placental form (GST-P)-positive preneoplastic lesions (p similar to<similar to 0.05) in the liver from TAA-treated groups. In conclusion, conventional coffee and 0.1% caffeine intake presented better beneficial effects than decaffeinated coffee against liver injury induced by TAA in male Wistar rats.

Serological detection of Plasmodium vivax malaria using recombinant proteins corresponding to the 19-kDa C-terminal region of the merozoite surface protein-1

Abstract Background Serological tests to detect antibodies specific to Plasmodium vivax could be a valuable tool for epidemiological studies, for screening blood donors in areas where the malaria is not endemic and for diagnosis of infected individuals. Because P. vivax cannot be easily obtained in vitro, ELISA assays using total or semi-purified antigens are rarely used. Based on this limitation, we tested whether recombinant proteins representing the 19 kDa C-terminal region of the merozoite surface protein-1 of P. vivax (MSP119) could be useful for serological detection of malaria infection. Methods Three purified recombinant proteins produced in Escherichia coli (GST-MSP119, His6-MSP119 and His6-MSP119-PADRE) and one in Pichia pastoris (yMSP119-PADRE) were compared for their ability to bind to IgG antibodies of individuals with patent P. vivax infection. The method was tested with 200 serum samples collected from individuals living in the north of Brazil in areas endemic for malaria, 53 serum samples from individuals exposed to Plasmodium falciparum infection and 177 serum samples from individuals never exposed to malaria. Results Overall, the sensitivity of the ELISA assessed with sera from naturally infected individuals was 95%. The proportion of serum samples that reacted with recombinant proteins GST-MSP119, His6-MSP119, His6-MSP119-PADRE and yMSP119-PADRE was 90%, 93.5%, 93.5% and 93.5%, respectively. The specificity values of the ELISA determined with sera from healthy individuals and from individuals with other infectious diseases were 98.3% (GST-MSP119), 97.7% (His6-MSP119 and His6-MSP119-PADRE) or 100% (yMSP119-PADRE). Conclusions Our study demonstrated that for the Brazilian population, an ELISA using a recombinant protein of the MSP119 can be used as the basis for the development of a valuable serological assay for the detection of P. vivax malaria.

Antioxidative responses of cell suspension cultures of two Coffea arabica varieties to low aluminum levels at pH 5.8

The effects of aluminum (Al) on the activities of antioxidant enzymes and ferritin expression were studied in cell suspension cultures of two varieties of Coffea arabica, Mundo Novo and Icatu, in medium with pH at 5.8. The cells were incubated with 300 µM Al3+, and the Al speciation as Al3+ was 1.45% of the mole fraction. The activities of superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST) were increased in Mundo Novo, whereas glutathione reductase (GR) and guaiacol peroxidase (GPOX) activities remained unchanged. SOD, GR, and GST activities were increased in Icatu, while CAT activity was not changed, and GPOX activity decreased. The expression of two ferritin genes (CaFer1 and CaFer2) were analyzed by Real-Time PCR. Al caused a downregulation of CaFER1 expression and no changes of CaFER2 expression in both varieties. The Western blot showed no alteration in ferritin protein levels in Mundo Novo and a decrease in Icatu. The differential enzymes responses indicate that the response to Al is variety-dependent.

Natural antibody response to Plasmodium falciparum merozoite antigens MSP5, MSP9 and EBA175 is associated to clinical protection in the Brazilian Amazon

BACKGROUND: Antibodies have an essential role in the acquired immune response against blood stage P. falciparum infection. Although several antigens have been identified as important antibody targets, it is still elusive which antigens have to be recognized for clinical protection. Herein, we analyzed antibodies from plasmas from symptomatic or asymptomatic individuals living in the same geographic area in the Western Amazon, measuring their recognition of multiple merozoite antigens. METHODS: Specific fragments of genes encoding merozoite proteins AMA1 and members of MSP and EBL families from circulating P. falciparum field isolates present in asymptomatic and symptomatic patients were amplified by PCR. After cloning and expression of different versions of the antigens as recombinant GST-fusion peptides, we tested the reactivity of patients' plasmas by ELISA and the presence of IgG subclasses in the most reactive plasmas. RESULTS: 11 out of 24 recombinant antigens were recognized by plasmas from either symptomatic or asymptomatic infections. Antibodies to MSP9 (X2(DF=1) = 9.26/p = 0.0047) and MSP5 (X2(DF=1) = 8.29/p = 0.0069) were more prevalent in asymptomatic individuals whereas the opposite was observed for MSP1 block 2-MAD20 (X2(DF=1) = 6.41/p = 0.0206, Fisher's exact test). Plasmas from asymptomatic individuals reacted more intensely against MSP4 (U = 210.5, p < 0.03), MSP5 (U = 212, p < 0.004), MSP9 (U = 189.5, p < 0.002) and EBA175 (U = 197, p < 0.014, Mann-Whitney's U test). IgG1 and IgG3 were predominant for all antigens, but some patients also presented with IgG2 and IgG4. The recognition of MSP5 (OR = 0.112, IC95% = 0.021-0.585) and MSP9 (OR = 0.125, IC95% = 0.030-0.529, cross tab analysis) predicted 8.9 and 8 times less chances, respectively, to present symptoms. Higher antibody levels against MSP5 and EBA175 were associated by odds ratios of 9.4 (IC95% = 1.29-69.25) and 5.7 (IC95% = 1.12-29.62, logistic regression), respectively, with an asymptomatic status. CONCLUSIONS: Merozoite antigens were targets of cytophilic antibodies and antibodies against MSP5, MSP9 and EBA175 were independently associated with decreased symptoms.