RESUMO
Four carbon materials, spent coffee-ground biochar, carbon black, short CNTs, and nitrogen-doped few-layer graphene (N-graphene) were tested for their functionalization with a commercial carboxylesterase. Their robustness to variations in time and key physicochemical parameters (temperature and pH) was analysed. In general, carbon nanomaterials showed better performance than biochar, both in terms of binding capacity and resilience in harsh conditions, at statistically significant levels. Among the tested materials, functionalized N-graphene also showed the highest level of inhibition of carboxylesterase by pesticide exposure. Therefore, N-graphene was selected for biotechnological application of pesticide scavenging toxicity in T. thermophila, a ciliate bioindicator of water quality. While immobilization of the enzyme was not effective in the case of carbaryl, a methyl carbamate, in the case of the organophosphorus dichlorvos, a 1- or 30-min contact time with a water solution containing 5 times the LC100 - 0.5 mM - allowed 50% and 100% rescue of ciliate survival, respectively. These results suggest that functionalization with carboxylesterase may be of additional benefit compared to bare carbon in water clean-up procedures, especially for highly hydrophilic pesticides such as dichlorvos.
Assuntos
Grafite , Nanoestruturas , Praguicidas , Praguicidas/toxicidade , Carboxilesterase/metabolismo , Carbaril , Diclorvós , Carbono , Biomarcadores Ambientais , Fuligem , Café , NitrogênioRESUMO
Breast reconstruction has gained from lipofilling the possibility to recover the aesthetic outcome of anatomical profile in a more natural appearance. However, until today, the long-term graft survival remains unpredictable, and sometimes it does not guarantee a well-adequate aesthetic result. In the present work, the morphological changes, occurring in fat mass used for refilling, harvested by the Coleman's procedure or through the washing/fragmenting procedure were analysed. Adipocyte size; immunohistochemistry against CD8, CD31, CD68 and M2-type macrophages and catalase enzyme, were analysed in vitro on fat mass cultured for 4 weeks. Our observation reveals an increase of connective tissue around the mass and a high number of immune cells occurrence in fat mass harvested by the Coleman's procedure. Instead, the washing/fragmented procedure would reduce the number of immune cells within the fat mass, increase the size of adipocytes, and cause a wider presence of active vessels profile and greater catalase expression. We hypothesize that the fat mass processed by the Coleman's procedure would remain more reactive due to a higher number of immune and macrophages cells, prone to develop cystic formation, leading to a suboptimal integration in the recipient site. On the other hand, the conditions more prone to realize an optimal integration would occur in the fat mass processed by the washing/fragmenting procedure: a reduced number of immune cells, low amount of connective tissue, presence of larger adipocytes. Follow-up monitoring did support our conclusion, as we observed a reduction of re-intervention for refilling procedure in patients treated with the washing/fragmenting procedure.
Assuntos
Tecido Adiposo , Coleta de Tecidos e Órgãos , Adipócitos , Humanos , Imuno-HistoquímicaRESUMO
BACKGROUND: Many pesticides have been shown to act as endocrine disrupters. Although the potencies of currently used pesticides as hormone agonists/antagonists are low compared with those of natural ligands, their ability to act via multiple mechanisms might enhance the biological effect. The organophosphate Chlorpyrifos (CHP) has been shown to be weakly estrogenic and cause adverse neurodevelopmental effects in mammals. However, no information is available on the endocrine effects of CHP in aquatic organisms. In the digestive gland of the bivalve Mytilus galloprovincialis, a target tissue of both estrogens and pesticides, the possible effects of CHP on the responses to the natural estrogen 17ß-estradiol (E(2)) were investigated. METHODOLOGY/PRINCIPAL FINDINGS: Mussels were exposed to CHP (4.5 mg/l, 72 hrs) and subsequently injected with E(2) (6.75 ng/g dw). Responses were evaluated in CHP, E(2) and CHP/E(2) treatment groups at 24 h p.i. by a biomarker/transcriptomic approach. CHP and E(2) induced additive, synergistic, and antagonistic effects on lysosomal biomarkers (lysosomal membrane stability, lysosome/cytoplasm volume ratio, lipofuscin and neutral lipid accumulation). Additive and synergistic effects were also observed on the expression of estrogen-responsive genes (GSTπ, catalase, 5-HTR) evaluated by RT-Q-PCR. The use of a 1.7K cDNA Mytilus microarray showed that CHP, E(2) and CHP/E(2), induced 81, 44, and 65 Differentially Expressed Genes (DEGs), respectively. 24 genes were exclusively shared between CHP and CHP/E(2), only 2 genes between E(2) and CHP/E(2). Moreover, 36 genes were uniquely modulated by CHP/E(2). Gene ontology annotation was used to elucidate the putative mechanisms involved in the responses elicited by different treatments. CONCLUSIONS: The results show complex interactions between CHP and E(2) in the digestive gland, indicating that the combination of certain pesticides and hormones may give rise to unexpected effects at the molecular/cellular level. Overall, these data demonstrate that CHP can interfere with the mussel responses to natural estrogens.
Assuntos
Biomarcadores/metabolismo , Clorpirifos/farmacologia , Sistema Digestório/efeitos dos fármacos , Estradiol/farmacologia , Inseticidas/farmacologia , Mytilus/efeitos dos fármacos , Mytilus/metabolismo , Animais , Sistema Digestório/metabolismo , Estrogênios/farmacologia , Perfilação da Expressão Gênica , Mytilus/crescimento & desenvolvimento , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The present research work was designed to study Dicentrarchus labrax biotransformation and detoxification responses to acute exposure to nickel (Ni) and chlorpyrifos (CHP). Sexually immature sea bass were treated by intraperitoneal injection of nickel chloride (500 µg kg⻹), chlorpyrifos (10 mg kg⻹), and their binary mixture for 1, 3, and 7 days. Ni and CHP accumulation was quantified in liver after the exposure periods. The following biological responses were measured: (1) NADPH cytochrome P450 reductase (NCR) activity, as phase I biotransformation parameter; (2) gluthathione S-transferase (GST) activity as a phase II conjugation enzyme, acetylcholinesterase activity, and metallothionein (MT) content. Ni bioaccumulation in the liver resulted in an increasing uptake up to 15.48 µg g⻹ wet weight (Ni-treated animals) and 16.73 µg g⻹ wet weight (mixture-treated animals) after 7 days of exposure. CHP accumulation showed a distinct pattern in animals exposed to the mixture of chemicals in comparison with CHP-treated animals. NCR activity exhibited a marked activation in CHP and mixture-treated animals. GST activity was significantly increased starting from 1 day exposure in CHP-treated animals and after 3 days in Ni-treated animals. MT accumulation increased in all conditions, with a marked synergetic effect after 7 days of exposure. These data should be carefully considered in view of the biological effects of mixture pollutants, particularly in fish farming conditions.
Assuntos
Bass/metabolismo , Clorpirifos/toxicidade , Níquel/toxicidade , Poluentes Químicos da Água/toxicidade , Acetilcolinesterase/metabolismo , Animais , Aquicultura , Clorpirifos/análise , Clorpirifos/metabolismo , Glutationa Transferase/metabolismo , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/metabolismo , Metalotioneína/metabolismo , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Níquel/análise , Níquel/metabolismo , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/metabolismoRESUMO
In the present study, mussel (Mytilus galloprovincialis) digestive gland oxidative stress biomarkers and detoxification responses to acute exposure to nickel (Ni) were investigated. Mussels were exposed to two sublethal concentrations of Ni (135 µg/L per animal (2.5 µM) and 770 µg/L per animal (13 µM)) for 24, 48, 72, 96 h and 8 days. Following biological responses were measured: (1) glutathione S-transferase (GST) activity as a phase II conjugation enzyme, (2) catalase activity as antioxidant response, (3) malondialdehyde accumulation (MDA) as lipid peroxydation marker and metallothionein as specific response to metals exposure. The cholinergic system was evaluated using the acetylcholinesterase activity (AChE). Moreover, Ni uptakes during the exposure periods were assessed and the uptake rate constant determined. A correlation matrix (CM) between the investigated biomarkers and a principal component analysis (PCA) were achieved for the two tested concentrations. The Ni-uptake constant was higher in animals exposed to the lowest concentration. The CM and the PCA showed a time-dependent effect of the Ni exposure on the investigated biomarkers being more pronounced in animals exposed to the highest Ni concentration. While AChE showed a significant increase after 48 h and a further return to control values in the lowest concentration, it was drastically maintained inhibited in the highest concentration. Our data provided clues about the occurrence of different toxicokinetics and toxicodynamics of two Ni sublethal concentrations in an ecologically relevant organism.
Assuntos
Poluentes Ambientais/farmacocinética , Poluentes Ambientais/toxicidade , Mytilus/metabolismo , Níquel/farmacocinética , Níquel/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Animais , Catalase/metabolismo , Mucosa Gástrica/metabolismo , Glutationa Transferase/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Malondialdeído/metabolismo , Metalotioneína/metabolismo , Mytilus/efeitos dos fármacos , Análise de Componente Principal , Fatores de TempoRESUMO
In this study, we report the tissue expression analysis of the p53 protein family mRNA in mussels (Mytilus spp.) by means of quantitative RT-PCR. The tissue specific response was evaluated after 24 h exposure to a sublethal benzo[a]pyrene (B[a]P) concentration (75 nM), showing a 2.6 fold induction in digestive gland cells and a dramatic gene down regulation in circulating hemocytes. The comet assay and DNA gel diffusion tests showed significant effects in hemocytes and negligible differences in the digestive gland nuclei, implicating p53 in DNA damage of molluscan hemocytes. Finally, the kinetics of p53 protein family mRNA expression in the digestive gland of animals exposed to B[a]P and crude oil (0.5 ppm) showed partially overlapping trends, characterised by a common down regulation after 1 week exposure. These data should be carefully considered in view of the biological effects of organic pollutants and particularly following spills.
Assuntos
Benzo(a)pireno/toxicidade , Mytilus/efeitos dos fármacos , Petróleo/toxicidade , RNA Mensageiro/metabolismo , Proteína Supressora de Tumor p53/genética , Poluentes Químicos da Água/toxicidade , Animais , Biomarcadores/metabolismo , Dano ao DNA , Sistema Digestório/efeitos dos fármacos , Sistema Digestório/metabolismo , Relação Dose-Resposta a Droga , Monitoramento Ambiental/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Hemócitos/efeitos dos fármacos , Hemócitos/metabolismo , Cinética , Mytilus/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Metallothioneins (MTs) are cysteine-rich proteins involved in homeostasis of essential metals, detoxification of toxic metals and scavenging of free radicals. Scavenging of the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical was measured by means of ESR spectroscopy for two recombinant MTs from aquatic species: MT-10 from the sea mussel Mytilus galloprovincialis, and MT-A from the fish Oncorhyncus mykiss. Both the zinc- and the cadmium-loaded forms (Zn(7)-MTs and Cd(7)-MTs) were analysed, using the commercial MT-II (Zn(7)-MT-II and Cd(7)-MT-II, respectively) from rabbit liver as a reference. A decrease in the scavenging ability was observed for all the three MTs passing from the Zn- to the Cd-loaded forms, because of the higher stability of the Cd-mercapto complex. The Zn(7)-MTs from aquatic species were more effective in scavenging DPPH signal than the rabbit Zn(7)-MT-II (2.8 and 4-folds, respectively). Similar results were obtained also for the Cd(7)-MTs, thus confirming the stronger antioxidant power of MTs from aquatic organisms compared with the rabbit MT-II. Moreover, mussel MT-10 was more active in DPPH scavenging than fish MT-A. When the complete release of metals from MTs was obtained by lowering the pH to 3 or, alternatively, by adding the chelating agent diethylenetriaminepentaacetic acid (DTPA), an increase in the scavenging ability of MTs was observed.
Assuntos
Sequestradores de Radicais Livres/farmacologia , Metalotioneína/fisiologia , Isoformas de Proteínas/fisiologia , Animais , Bivalves , Espectroscopia de Ressonância de Spin Eletrônica , PeixesRESUMO
The effect of growth hormone (GH) and cadmium (Cd) on metallothionein (MT) expression was investigated in hepatoma cells. In fish the constitutive isoform MT-B and the metal-responsive MT-A are expressed. Real-time RT-PCR revealed that: Cd up-regulates mostly MT-A, GH slightly induces MT-B and the GH/Cd combination induces synergistically both MTs. Perturbations in Ca2+ levels suppressed or reduced the Cd-induction of MTs and abolished the GH/Cd synergy. Similar results were obtained by inhibition of tyrosine kinases. Also the signaling molecules recruited by the GH receptor responded differently to GH and Cd, with ERKs showing a synergistic activation upon GH/Cd. The following conclusions can be drawn: (1) cytosolic Ca2+ is mainly involved in MT-A regulation; (2) both Ca2+ and tyrosine phosphorylation are essential for Cd-induction and GH/Cd synergy on MTs. The synergy could depend on interactions in different signaling pathways, leading to a differential recruitment of MTF-1 and AP-1 transcription factors.
Assuntos
Cádmio/farmacologia , Carcinoma Hepatocelular/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Hormônio do Crescimento/farmacologia , Metalotioneína/genética , Animais , Cálcio/metabolismo , Carcinoma Hepatocelular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Sinergismo Farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Metalotioneína/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oncorhynchus mykiss/metabolismo , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores da Somatotropina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Células Tumorais Cultivadas , Tirosina/metabolismo , Fator MTF-1 de TranscriçãoRESUMO
A combination of biomarkers and gene expression analyses was used to investigate the occurrence of a stress syndrome in mussels (Mytilus edulis) caged along a copper pollution gradient in the Visnes fjord, Norway. The stress level in mussels, as calculated by a novel algorithm (the "Expert System") from a set of seven biomarkers, was compared with gene expression changes utilising a low-density oligonucleotide microarray, employing 24 different genes involved in both cellular homeostasis and stress-related responses. The biomarker battery included lysosomal membrane stability, lysosomal accumulation of neutral lipids and lipofuscin, lysosomal/cytoplasm volume ratio, Ca(2+)-ATPase and catalase activities, and total metallothionein content. Integration of the biomarkers into the Expert System ranked individuals sampled at site 2 as unstressed, mussels sampled at site 3 as being subject to low stress, and those from site 4, which is adjacent to what used to be a copper mine, as being highly stressed, with respect to specimens sampled from the reference site. Microarray analyses demonstrated that at the two innermost and mostly polluted sites, gene expression patterns where severely altered. In particular, some genes exhibited a linear activation response along the copper gradient, e.g. metallothioneins mt 20 and mt 10, and catalase. In addition, stress responsive kinase (krs), glutathione transferase (gst), major vault protein and histones (h1, h2a and h4) were significantly up-regulated at the innermost site. In conclusion, these results demonstrated that sites could be discriminated using both a physiological and a molecular approach. The development of a stress syndrome along the pollution gradient was evidenced with a novel mussel microarray, both in terms of numbers of regulated genes and level of gene response.
Assuntos
Cobre/toxicidade , Exposição Ambiental , Lisossomos/efeitos dos fármacos , Mytilus edulis/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Biomarcadores/análise , ATPases Transportadoras de Cálcio/efeitos dos fármacos , ATPases Transportadoras de Cálcio/metabolismo , Catalase/efeitos dos fármacos , Catalase/metabolismo , Primers do DNA/química , Perfilação da Expressão Gênica/veterinária , Glutationa Transferase/efeitos dos fármacos , Lipídeos/análise , Lipofuscina/análise , Lisossomos/enzimologia , Lisossomos/metabolismo , Metalotioneína/análise , Metalotioneína/genética , Metalotioneína/fisiologia , Mytilus edulis/enzimologia , Mytilus edulis/fisiologia , Noruega , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Regulação para CimaRESUMO
We have studied the effects of heavy metals (Hg2+, Cu2+, Cd2+) on growth hormone (GH) activation of tyrosine kinase and Ca2+ signaling in the trout (Oncorhynchus mykiss) hepatoma cell line RTH-149. Molecular cloning techniques using primer designed on Oncorhynchus spp. growth hormone receptor (GHR) genes allowed to isolate a highly homologous cDNA fragment from RTH-149 mRNA. Thereafter, cells were analysed by Western blotting or, alternatively, with Ca2+ imaging using fura-2/AM. Exposure of cells to ovine GH alone produced a stimulation of the JAK2/STAT5 pathway and intracellular free Ca2+ variations similar to what has been observed in mammalian models. Cell pre-exposure to Cu2+, Hg2+ or Cd2+ affected cell response to GH by enhancing (Cu2+) or inhibiting (Cd2+) the phosphorylation of JAK2 and STAT5. Heavy metals induced the activation of the MAP kinase p38, and pre-exposure to Hg2+ or Cu2+ followed by GH enhanced the effect of metal alone. Image analysis of fura2-loaded cells indicated that pre-treatment with Hg2+ prior to GH produced a considerable increase of the [Ca2+]i variation produced by either element, while using Cu2+ or Cd2+ the result was similar but much weaker. Data suggest that heavy metals interfere with GH as follows: Hg2+ is nearly ineffective on JAK/STAT and strongly synergistic on Ca2+ signaling; Cu2+ is activatory on JAK/STAT and slightly activatory on Ca2+; Cd2+ is strongly inhibitory on JAK/STAT and slightly activatory on Ca2+; heavy metals could partially activate STAT via p38 independently from GH interaction.
Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Hormônio do Crescimento/farmacologia , Metais Pesados/toxicidade , Receptores da Somatotropina/genética , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Carcinoma Hepatocelular , Linhagem Celular Tumoral , Modelos Animais de Doenças , Neoplasias Hepáticas , Dados de Sequência Molecular , Oncorhynchus mykiss , RNA Mensageiro/genética , Receptores da Somatotropina/química , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais/efeitos dos fármacosRESUMO
Mercury is a non-essential heavy metal affecting intracellular Ca2+ dynamics. We studied the effects of Hg2+ on [Ca2+]i in trout hepatoma cells (RTH-149). Confocal imaging of fluo-3-loaded cells showed that Hg2+ induced dose-dependent, sustained [Ca2+]i transient, triggered intracellular Ca2+ waves, stimulated Ca2+-ATPase activity, and promoted InsP3 production. The effect of Hg2+ was reduced by the Ca2+ channel blocker verapamil and totally abolished by extracellular GSH, but was almost unaffected by cell loading with the heavy metal chelator TPEN or esterified GSH. In a Ca2+-free medium, Hg2+ induced a smaller [Ca2+]i transient, that was unaffected by TPEN, but was abolished by U73122, a PLC inhibitor, and by cell loading with GDP-betaS, a G protein inhibitor, or heparin, a blocker of intracellular Ca2+ release. Data indicate that Hg2+ induces Ca2+ entry through verapamil-sensitive channels, and intracellular Ca2+ release via a G protein-PLC-InsP3 mechanism. However, in cells loaded with heparin and exposed to Hg2+ in the presence of external Ca2+, the [Ca2+]i rise was maximally reduced, indicating that the global effect of Hg2+ is not a mere sum of Ca2+ entry plus Ca2+ release, but involves an amplification of Ca2+ release operated by Ca2+ entry through a CICR mechanism.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Glutationa/análogos & derivados , Guanosina Difosfato/análogos & derivados , Mercúrio/toxicidade , Compostos de Anilina/análise , Compostos de Anilina/farmacocinética , Animais , ATPase de Ca(2+) e Mg(2+)/metabolismo , Cálcio/análise , Sinalização do Cálcio/efeitos dos fármacos , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/fisiopatologia , Linhagem Celular Tumoral/efeitos dos fármacos , Citosol/química , Citosol/efeitos dos fármacos , Citosol/metabolismo , Estrenos/farmacologia , Etilenodiaminas/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Glutationa/farmacologia , Guanosina Difosfato/farmacologia , Heparina/farmacologia , Histocitoquímica , Inositol 1,4,5-Trifosfato/análise , Inositol 1,4,5-Trifosfato/metabolismo , Cinética , Mercúrio/farmacologia , Microscopia Confocal , Microscopia de Fluorescência , Fosfolipases/metabolismo , Pirrolidinonas/farmacologia , Tionucleotídeos/farmacologia , Truta , Verapamil/farmacologia , Xantenos/análise , Xantenos/farmacocinéticaRESUMO
Metallothionein (MT) is a ubiquitous, metal-inducible protein with an important role in the homeostasis and in the detoxification of heavy metals. This work reports the cloning and sequencing of a MT gene encoding a MT isoform (MT20-IIIa) in the mussel Mytilus galloprovincialis Lam, a lamellibranch mollusc known to accumulate and to detoxify large amounts of metal. The MT gene, lacking the 5' promoter region, is 1865 bp long and has a tripartite structure consisting of three exons and two introns. The putative open reading frame (ORF) encodes a polypeptide of 72 amino acids, which corresponds to the MT-I class, type 2 family (http://www.unizh.ch/~mtpage/classif.html). The structure of the gene and the putative MT20-III protein have been compared with those of other species. The putative biological significance of the differences at the amino acid level among the different MTs is discussed.
Assuntos
Clonagem Molecular , Metalotioneína/genética , Análise de Sequência de DNA , Sequência de Aminoácidos , Animais , Sequência de Bases , Bivalves , Éxons , Íntrons , Dados de Sequência Molecular , Fases de Leitura Aberta , Isoformas de Proteínas/genética , Homologia de Sequência de AminoácidosRESUMO
Dictyostelium discoideum amoebae showed an uncommon resistance to Cu(2+), as pointed out through cell growth rate (EC(50) = 469 +/- 30 microM) and the neutral red cytotoxicity assay (EC(50) = 334 +/- 45 microM). Although no evidence of Cu-inducible metallothionein was found, Cu-dependent ATPase activity was cytochemically detected on pelletted, resin-embedded amoebae. This activity required Cu(2+) in the incubation medium, was sensitive to TPEN, vanadate and temperature, and showed dose-dependent increase after exposure of amoebae to 10-500 microM Cu(2+) for 7 days. Accordingly, immunofluorescence and Western blotting revealed the occurrence of a Cu-inducible, putative homologue of human Menkes (MNK) Cu-P-type ATPase. To verify if Cu-ATPase is involved in copper resistance, amoebae were exposed to low concentrations of Cu(2+) and vanadate followed by the neutral red assay. Exposure to either treatment showed no effect, while a combination caused a dramatic increase of Cu toxicity, possibly depending on Cu-ATPase inhibition.