[show abstract][hide abstract] ABSTRACT: Fish bile metallothioneins (MT) have been recently reported as biomarkers for environmental metal contamination; however, no studies regarding standardizations for their purification are available. Therefore, different procedures (varying centrifugation times and heat-treatment temperatures) and reducing agents (DTT, β-mercaptoethanol and TCEP) were applied to purify MT isolated from fish (Oreochromis niloticus) bile and liver. Liver was also analyzed, since these two organs are intrinsically connected and show the same trend regarding MT expression. Spectrophotometrical analyses were used to quantify the resulting MT samples, and SDS-PAGE gels were used to qualitatively assess the different procedure results. Each procedure was then statistically evaluated and a multivariate statistical analysis was then applied. A response surface methodology was also applied for bile samples, in order to further evaluate the responses for this matrix. Heat treatment effectively removes most undesired proteins from the samples, however results indicate that temperatures above 70°C are not efficient since they also remove MTs from both bile and liver samples. Our results also indicate that the centrifugation times described in the literature can be decreased in order to analyze more samples in the same timeframe, of importance in environmental monitoring contexts where samples are usually numerous. In an environmental context, biliary MT was lower than liver MT, as expected, since liver accumulates MT with slower detoxification rates than bile, which is released from the gallbladder during feeding, and then diluted by water. Therefore, bile MT seems to be more adequate in environmental monitoring scopes regarding recent exposure to xenobiotics that may affect the proteomic and metalloproteomic expression of this biological matrix.
[show abstract][hide abstract] ABSTRACT: The pacu (Piaractus mesopotamicus) is a hypoxia-tolerant neotropical fish species. There is little or no information in this species regarding biochemical adaptations to waters with different oxygen concentrations, such as the production of reactive oxygen species and antioxidant scavengers, which might be of interest in the study of antioxidant defense mechanisms. Metallothioneins (MT) have been widely applied as biomarkers for metal exposure in fish liver, and, recently, in bile. These metalloproteins, however, have also been reported as free radical scavengers, although studies in this regard are scarce in fish. In this context, normoxic and hypoxic controlled experiments were conducted with pacu specimens and MT levels were quantified in both liver and bile. Reduced glutathione (GSH) indicative of oxidative stress, and thiobarbituric acid reactive substances (TBARS), indicative of lipid peroxidation, were also determined in liver. The results demonstrate that hypoxic fish present significantly lower metallothionein levels in liver and bile and lower reduced glutathione levels in liver, whereas lipid peroxidation was not significantly different between hypoxic and normoxic fish. The results of the present study seem to suggest that metallothioneins may actively participate in redox regulation in hypoxic fish in both bile and liver. MT levels in these organs may be temporarily suppressed, supporting the notion that down-regulation of oxidant scavengers during the oxidative burst is important in defense signaling in these adapted organisms.
Ecotoxicology and Environmental Safety 02/2014; 103C:24-28. · 2.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: The present study analyzed metallothionein (MT) excretion from liver to bile in Nile Tilapia (Oreochromis niloticus) exposed to sub-lethal copper concentrations (2mgL(-1)) in a laboratory setting. MTs in liver and bile were quantified by spectrophotometry after thermal incubation and MT metal-binding profiles were characterized by size exclusion high performance liquid chromatography coupled to ICP-MS (SEC-HPLC-ICP-MS). Results show that liver MT is present in approximately 250-fold higher concentrations than bile MT in non-exposed fish. Differences between the MT profiles from the control and exposed group were observed for both matrices, indicating differential metal-binding behavior when comparing liver and bile MT. This is novel data regarding intra-organ MT comparisons, since differences between organs are usually present only with regard to quantification, not metal-binding behavior. Bile MT showed statistically significant differences between the control and exposed group, while the same did not occur with liver MT. This indicates that MTs synthesized in the liver accumulate more slowly than MTs excreted from liver to bile, since the same fish presented significantly higher MT levels in liver when compared to bile. We postulate that bile, although excreted in the intestine and partially reabsorbed by the same returning to the liver, may also release MT-bound metals more rapidly and efficiently, which may indicate an efficient detoxification route. Thus, we propose that the analysis of bile MTs to observe recent metal exposure may be more adequate than the analysis of liver MTs, since organism responses to metals are more quickly observed in bile, although further studies are necessary.
Journal of Trace Elements in Medicine and Biology 09/2013; · 1.96 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mullet (Mugil liza) were sampled in five different areas along the Guanabara Bay, southeastern Brazil, classified as non-contaminated, moderately contaminated and contaminated. Morphometric (Fulton condition factor, relative condition factor and weight to length scaling coefficient) and organosomatic (hepatosomatic index) indices of environmental stress were analysed. Fish from the differentially contaminated areas show statistically different Fulton and relative condition factors and hepatosomatic indices, but not the weight to length scaling coefficient. The Kn and the FCF followed the same trend, with fish from São Gonçalo (1.07 ± 0.04 and 0.89 ± 0.03), Itaipu (0.84 ± 0.01 and 0.86 ± 0.01) and the Rodrigo de Freitas Lagoon (1.03 ± 0.01 and 0.87 ± 0.20) showing higher FCFs than fish from Magé (0.96 ± 0.01 and 0.81 ± 0.01). Fish from Itaipu showed significantly higher HSI values than the other sampling sites (1.68 ± 0.07), with fish from Olaria and Ipiranga showing the lowest (1.56 ± 0.12 and 1.60 ± 0.07, respectively).
Bulletin of Environmental Contamination and Toxicology 10/2012; · 1.11 Impact Factor
[show abstract][hide abstract] ABSTRACT: Glutathione S-transferase (GST) assays in non-mammalian organisms are usually conducted inappropriately, since no previous standardization of the optimal concentrations of proteins and substrates and adequate pH is conducted. Standardization is a key task to adjust enzyme assays at their kinetically correct maximal initial velocities, if one wants these velocities to indicate the amount of enzyme in a sample. In this paper GST assays were standardized in liver cytosol to compare seasonal GST levels in liver of mullet from two contaminated lagoons in the Rio de Janeiro to those from a reference bay. GST potential as a biomarker of sublethal intoxication in this species was also evaluated. Mullet liver GST levels assayed with substrates that corresponded to three different GST isoenzymes varied throughout the year. The differences indicated that mullets are suffering from sublethal intoxication from contaminants in these lagoons. Seasonal variations of activity were relevant, since these could indicate differences in xenobiotic input into the areas. An analysis of overall mullet health condition using a morphological index (the Fulton Condition Factor) and macroscopic abnormalities corroborated the differences in GST levels, with fish from one of the sites in worse overall health condition showing lower and significantly different FCF when compared to the reference site. Therefore, GST standardized activity levels are useful biomarkers of environmental contamination for mullet.
[show abstract][hide abstract] ABSTRACT: Fish accumulate several trace elements in muscle, gills and liver, however studies also indicate that metals can be excreted through bile. Since metal contamination leads to modifications in bile composition, biliary excretion offers an alternative way to evaluate the presence of trace-elements. Bile is easier to obtain than other organs and presents a simpler matrix, making it easier for chemical pre-treatment. To verify if bile can be useful as a biomonitoring tool for metal contamination, liver and bile trace element concentrations were determined and correlated. The Artificial Neural Networks statistical technique was used to verify if liver trace-element quantification could be substituted by bile analysis. Results show that significant correlations were obtained between trace elements in bile and liver and the ANN validated the hypothesis that certain trace-elements in bile could be utilized instead of liver trace-elements. Further studies in this field are of interest to further validate this biomarker.
[show abstract][hide abstract] ABSTRACT: Gallbladder bile from 2 fish species, mullet (Mugil liza) and tilapias (Tilapia rendalli), contain substantial matrix metalloproteinases (MMPs). Extensive purification studies were conducted in order to obtain workable samples for SDS-PAGE and zymography analysis. Proteinase activities were assayed by gelatin substrate zymography. Several protein bands were observed, corresponding to molecular weights of 200, 136, 43, 36, 34, 29, 23 and 14 kDa in mullet bile and 179, 97, 79, 61, 54, 45, 36, 33 and 21 kDa in tilapia bile. Specific inhibitor studies were conducted, in which MMPS were inhibited by EDTA and 1,10 phenanthroline, but not by serine and cysteine protease inhibitors, such as phenylmethylsulfonyl fluoride (PMSF) and transepoxysuccinyl-l-leucylamido-l-guanidino butane (E-64), confirming the proteinase identities as MMPs. Differences in proteinase expression were observed in fish from a contaminated and reference site. Some studies regarding MMPs in different fish tissues exist, however this is the first study conducted in fish bile, and their involvement in detoxification processes and organism protection against the effects of aquatic contaminants may be a possibility.
[show abstract][hide abstract] ABSTRACT: Metallothioneins (MT) are a validated biomarker for metal exposure. In fish they are usually determined in the liver, while some studies have observed MT in kidney, muscle and gills. There are, however, no reports on MT in fish bile. This study aimed to verify if these proteins are present in fish bile, if they can be used as biomarkers for metal exposure and to characterize the metal speciation present in the different species. Three independent methods to verify MT presence in fish bile were conducted: 1D/2D gel electrophoresis, spectrophotometric analyses and SEC-HPLC-ICP-MS. Results show that all methods verified the presence of these proteins in fish bile, while SEC-HPLC-ICP-MS also characterized also characterized metals responsible for the contamination of the studied sites. Quantification was successfully conducted by two methods, densitometry (1D electrophoresis) and spectrophotometry, and the data indicates that bile MTs follow the same trend as in liver. Therefore, Biliary MT show the potential to be used the same way as MT from other matrices in metal exposure studies, with the advantage of being a simpler matrix and showing the possibility of not sacrificing the animals, as chronic duct cannulation is available.
[show abstract][hide abstract] ABSTRACT: Fish are well-recognized bioindicators of environmental contamination. Several recent proteomic studies have demonstrated the validity and value of using fish in the search and discovery of new biomarkers. Certain analytical tools, such as comparative protein expression analyses, both in field and lab exposure studies, have been used to improve the understanding of the potential for chemical pollutants to cause harmful effects. The metallomic approach is in its early stages of development, but has already shown great potential for use in ecological and environmental monitoring contexts. Besides discovering new metalloproteins that may be used as biomarkers for environmental contamination, metallomics can be used to more comprehensively elucidate existing biomarkers, which may enhance their effectiveness. Unfortunately, metallomic profiling for fish has not been explored, because only a few fish metalloproteins have thus far been discovered and studied. Of those that have, some have shown ecological importance, and are now successfully used as biomarkers of environmental contamination. These biomarkers have been shown to respond to several types of environmental contamination, such as cyanotoxins, metals, and sewage effluents, although many do not yet possess any known function. Examples of successes include MMPs, superoxide dismutases, selenoproteins, and iron-bound proteins. Unfortunately, none of these have, as yet, been extensively studied. As data are developed for them, valuable new information on their roles in fish physiology and in inducing environmental effects should become available.
Reviews of environmental contamination and toxicology 01/2012; 218:101-23. · 4.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: A new zinc(II) complex of the mycobactericidal drug isoniazid (complex 1) was synthesized and characterized by XRD, vibrational spectroscopy (IR, Raman) and thermogravimetric analysis. The complex is constituted by two isoniazid (INH) molecules, six hydration water molecules and two perchlorate counter-ions for each metal center (C12H26N6Cl2O16Zn). Zinc(II) adopts a distorted octahedral geometry, where two INH molecules coordinate in a bidentate manner through the hydrazide group (N, O) and the other two isoniazid residues complete the coordination sphere of zinc(II) through their aromatic nitrogen atoms. This coordination pattern gives rise to a 2-D coordination polymer. Complex 1 belongs to the monoclinic system [a=8.1190(2)Å, b=17.977(4)Å, c=9.1051(2)Å and β=100.87(3)°], space group P21. A biological assay with Artemia salina was also performed. Complex 1 is almost 8.5 times more active than the free ligand. Its toxicity against A. salina correlates well with the cytotoxic activity for some human solid tumors. Therefore, antitumoral properties could be expected from complex 1.
[show abstract][hide abstract] ABSTRACT: This study investigated pyrene adsorption on two contrasting Brazilian soils: a Kandiudult and a Vertisol. It was found that
the time taken to reach thermodynamic equilibrium depended on the soil type. The curves for different pyrene-to-soil mass
ratios for Vertisol soil showed significant differences. This is probably related to the presence of 2:1 clays that may increase
the adsorption of pyrene due to the resulting interlamellar space. The adsorption of pyrene on the Kandiudult showed, in general,
good agreement with the Langmuir isotherm. In the case of the Vertisol, there was good agreement with the linear isotherm.
The kinetic model that best explains the adsorption in Kandiudult was the pseudo second-order model. For the Vertisol, the
Morris Weber model best explains the behavior of pyrene.
Water Air and Soil Pollution 01/2011; 219(1):297-301. · 1.75 Impact Factor
[show abstract][hide abstract] ABSTRACT: On April 26th, 2005, an accident caused a leak of 60,000L of Diesel Oil Type "B", freighted by train wagons upstream on a mangrove area within Guanabara Bay, Southeast Brazil. After the accident, samples from animals with different biological requirements were collected in order to monitor polycyclic aromatic hydrocarbons concentrations for the following 12months. Sessile, mobile, carnivorous, omnivorous, organic detritus feeders, planktivorous and suspension feeders were some of the attributes compared. Concentrations of PAHs did not vary in relation to different dietary habits and the best response was from the sessile suspensivorous barnacles. A background level of <50microgkg(-1) was suggested based on the reference site and on values observed in the following months after the accident. The highest values of PAH concentrations were observed in barnacles in the first month immediately after the spill, decreasing to background levels after few months. Barnacles are suggested as a sentinel species.
[show abstract][hide abstract] ABSTRACT: This study used the Discriminant Analysis statistical technique and Artificial Neural Networks, multilayer perceptron, in the classification of three fish species sampled in the state of Rio de Janeiro, Brazil: Geophagus brasiliensis (acaras), Tilapia rendall (tilapias) and Mugil liza (mullets). These fish were sexed when possible, weighed, measured, and had their Gonadosomatic and Hepatosomatic Indices calculated, as well as their Condition Factor. The use of an Artificial Neural Network (ANN) presented satisfactory results, even though the groups were composed of very diverse-sized animals. Without the need for non-violation assumptions and other considerations, the Artificial Neural Network was found to be the excellent alternative to classification problems of unbalanced data, such as the one presented in this study.
[show abstract][hide abstract] ABSTRACT: Iron(III) complexes [Fe(H2Fo4pT)Cl3] (1), [Fe(H2Ac4pT)Cl3] (2) and [Fe(H2Bz4pT)Cl3] (3) with N4 -para-tolyl-thiosemicarbazones derived from 2-formyl (H2Fo4pT), 2-acetyl (H2Ac4pT) and 2-benzoylpyridine (H2Bz4pT) were prepared and characterized. EPR data for 1–3 reveal the presence of low-spin iron(III) with d xz ²d yz ²d xy ¹ ground state. Electrochemical studies of the complexes showed mostly metal-centered redox changes with a quasi-reversible Fe(III)/Fe(II) couple. H2Fo4pT and H2Ac4pT exhibited toxicity against Artemia salina at low doses (LD50 = 27.5 µM and LD50 = 4.7 µM, respectively). Upon coordination the toxicity increased substantially in the case of [Fe(H2Fo4pT)Cl3] (LD50 = 1.9 µM) and did not change for [Fe(H2Ac4pT)Cl3]. H2Bz4pT and its iron(III) complex were not soluble in water.
Journal of Coordination Chemistry. 03/2009; 62(6):958-966.
[show abstract][hide abstract] ABSTRACT: A careful study was performed to evaluate and compare uncertainties associated with the measurement of luminescence from chrysene by using fluorimetry in solution and by using solid surface room-temperature phosphorimetry (SSRTP). Chrysene was chosen as the luminescent analyte (measurand) for this study because it is a substance of environmental interest and also because it presents strong natural fluorescence and phosphorescence that is easily induced by the external heavy atom effect. The most common approach for uncertainty calculation is the one indicated in the Guide to the Expression of Uncertainty in Measurement which is recognized worldwide. The most relevant sources of uncertainty were identified. The expanded uncertainty (k = 2; 95%) for SSRTP was 3.27 × 10−6 mol L−1 (3.7 ng of chrysene) with relevant contributions from the internal reproducibility, preparation of solutions and from the analytical curve. This value is equivalent to 28% of the reference analyte value. For fluorimetry, an acceptable value for the expanded uncertainty was achieved by preparing solutions by weighing masses of analyte and solvent. In this case, the huge contribution from the preparation of solutions by adjusting volume is minimized and the uncertainty magnitude was 2.12 × 10−9 mol L−1 (21.2% of the chrysene reference concentration). In this case, relevant contributions were from the repeatability, the internal reproducibility and from the analytical curve. The preparation of solutions by weighing does not have a relevant impact in improving the quality of the signal measurements by SSRTP.
[show abstract][hide abstract] ABSTRACT: The iron(III) complexes [Fe(2Am4DH)2]Cl (1), [Fe(2Am4Me)2]Cl (2) and [Fe(2Am4Et)2]Cl (3) with 2-pyridineformamide thiosemicarbazone (H2Am4DH) and its N(4)-methyl (H2Am4Me) and N(4)-ethyl (H2Am4Et) derivatives were obtained and characterized by means microanalyses, infrared and EPR spectra. The electrochemical behavior of the complexes was investigated. The thiosemicarbazones and complexes (1) and (2) presented toxicity against Artemia salina at low concentrations. Since this bioassay has a good correlation with cytotoxic activity in human solid tumors, the studied compounds present potential pharmacological applications.Graphical abstractThe iron(III) complexes [Fe(2Am4DH)2]Cl (1), [Fe(2Am4Me)2]Cl (2) and [Fe(2Am4Et)2]Cl (3) with 2-pyridineformamide thiosemicarbazone (H2Am4DH) and its N(4)-methyl (H2Am4Me) and N(4)-ethyl (H2Am4Et) derivatives were obtained. The thiosemicarbazones and complexes 1 and 2 presented toxicity against Artemia salina at low concentrations, suggesting that the studied compounds might present antineoplastic activity. View Within Article