RESUMEN

Despite the fact that environmental pollution has been implicated in the global rise of diabetes, the research on the impact of emerging pollutants such as novel flame retardants remains limited. In line with the shift towards the use of non-animal approaches in toxicological testing, this study aimed to investigate the effects of two novel flame retardants tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) and triphenyl phosphate (TPhP) in rat (INS1E) and human (NES2Y) pancreatic beta-cell lines. One-week exposure to 1 µM and 10 µM TDCIPP and TPhP altered intracellular insulin and proinsulin levels, but not the levels of secreted insulin (despite the presence of a statistically insignificant trend). The exposures also altered the protein expression of several factors involved in beta-cell metabolic pathways and signaling, including ATP citrate lyase, isocitrate dehydrogenase 1, perilipins, glucose transporters, ER stress-related factors, and antioxidant enzymes. This study has brought new and valuable insights into the toxicity of TDCIPP and TPhP on beta-cell function and revealed alterations that might impact insulin secretion after more extended exposure. It also adds to the scarce studies using in vitro pancreatic beta-cells models in toxicological testing, thereby promoting the development of non-animal testing strategy for identifying pro-diabetic effects of chemical pollutants.

RESUMEN

Decades after most countries banned hexachlorocyclohexane, HCH isomers still pollute the environment. Many studies described HCH as a pro-diabetic factor; nevertheless, the effect of HCH isomers on pancreatic beta-cells remains unexplored. This study investigated the effects of a one-month exposure to α-HCH, ß-HCH, and γ-HCH on protein expression in human (NES2Y) and rat (INS1E) pancreatic beta-cell lines. α-HCH and γ-HCH increased proinsulin and insulin levels in INS1E cells, while ß-HCH showed the opposite trend. α-HCH altered the expression of PKA, ATF3, and PLIN2. ß-HCH affected the expression of GLUT1, GLUT2, PKA, ATF3, p-eIF2α, ATP-CL, and PLIN2. γ-HCH altered the expression of PKA, ATF3, PLIN2, PLIN5, and IDH1. From the tested proteins, PKA, ATF3, and PLIN-2 were the most sensitive to HCH exposure and have the potential to be used as biomarkers.

Asunto(s)

Contaminantes Ambientales , Hexaclorociclohexano , Humanos , Animales , Ratas , Hexaclorociclohexano/toxicidad , Hexaclorociclohexano/metabolismo , Contaminantes Ambientales/toxicidad , Isomerismo

RESUMEN

Caffeic acid belongs to the polyphenol compounds we consume daily, often in the form of coffee. Even though it is less explored than caffeic acid phenethyl ester, it still has many positive effects on human health. Caffeic acid can affect cancer, diabetes, atherosclerosis, Alzheimer's disease, or bacterial and viral infections. This review focuses on the molecular mechanisms of how caffeic acid achieves its effects.

Asunto(s)

Ácidos Cafeicos , Alcohol Feniletílico , Humanos , Ácidos Cafeicos/farmacología , Alcohol Feniletílico/farmacología

RESUMEN

Many compounds have the potential to harm pancreatic beta-cells; organochlorine pollutants belong to those compounds. In this work, we aimed to find markers of acute toxicity of p,p'-DDT exposure among proteins expressed in NES2Y human pancreatic beta-cells employing 2-D electrophoresis. We exposed NES2Y cells to a high concentration (150 µM, LC96 after 72 hours) of p,p'-DDT for 24 and 30 hours and determined proteins with changed expression using 2-D electrophoresis. We have found 22 proteins that changed their expression. They included proteins involved in ER stress (GRP78, and endoplasmin), mitochondrial proteins (GRP75, ECHM, IDH3A, NDUS1, and NDUS3), proteins involved in the maintenance of the cell morphology (EFHD2, TCPA, NDRG1, and ezrin), and some other proteins (HNRPF, HNRH1, K2C8, vimentin, PBDC1, EF2, PCNA, biliverdin reductase, G3BP1, FRIL, and HSP27). The proteins we have identified may serve as indicators of p,p'-DDT toxicity in beta-cells in future studies, including long-term exposure to environmentally relevant concentrations.

Asunto(s)

Biomarcadores/metabolismo , DDT/toxicidad , Células Secretoras de Insulina/citología , Proteómica/métodos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Electroforesis en Gel Bidimensional , Chaperón BiP del Retículo Endoplásmico , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Espectrometría de Masas

RESUMEN

Persistent organochlorine pollutants (POPs) gradually accumulate in the human organism due to their presence in the environment. Some studies have described a correlation between the level of POPs in the human body and the incidence of diabetes, but we know little about the direct effect of POPs on pancreatic beta-cells. We exposed pancreatic beta-cells INS1E to non-lethal concentrations of p,p'-DDT (1,1'-(2,2,2-Trichloroethane-1,1-diyl)bis(4-chlorobenzene)) and p,p'-DDE (1,1'-(2,2-dichloroethene-1,1-diyl)bis(4-chlorobenzene)) for 1 month, and assessed changes in protein expression and the intracellular insulin level. 2-D electrophoresis revealed 6 proteins with changed expression in cells exposed to p,p'-DDT or p,p'-DDE. One of the detected proteins - vitamin D-binding protein (VDBP) - was upregulated in both cells exposed to p,p'-DDT, and cells exposed to p,p'-DDE. Both exposures to pollutants reduced the intracellular level of insulin mRNA, proinsulin, and insulin monomer; p,p'-DDT also slightly reduced the level of hexameric insulin. Overexpression of VDBP caused by the stable transfection of beta-cells with the gene for VDBP decreased both the proinsulin and hexameric insulin level in beta-cells similarly to the reduction detected in cells exposed to p,p'-DDT. Our data suggest that in the cells exposed to p,p'-DDT and p,p'-DDE, the increased VDBP protein level decreased the proinsulin expression in an unknown mechanism.

Asunto(s)

Contaminantes Ambientales/toxicidad , Células Secretoras de Insulina/efectos de los fármacos , Insulina/metabolismo , Proteína de Unión a Vitamina D/metabolismo , Animales , Línea Celular , DDT/toxicidad , Diclorodifenil Dicloroetileno/toxicidad , Células Secretoras de Insulina/metabolismo , Ratas , Pruebas de Toxicidad Subcrónica , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

PURPOSE: Lipotoxicity is implicated in type 2 diabetes pathogenesis. Its molecular mechanisms are not completely understood. The aim of this study is to identify new suspect proteins involved in pancreatic ß-cell death induction by saturated fatty acids and its inhibition by unsaturated fatty acids. EXPERIMENTAL DESIGN: Employing 2DE analysis and subsequent western blot confirmation, the differences in membrane/membrane-associated protein expression in human ß-cell line NES2Y are assessed during cell death induction by stearate and its inhibition by oleate. RESULTS: Induction of apoptosis by stearate is associated with significantly increased levels of Hsp90ß, peroxiredoxin-1, and 14-3-3γ in the membrane fraction of NES2Y cells and significantly decreased levels of annexin A2, annexin A4, and reticulocalbin-2. All these changes are significantly inhibited by oleate co-application. No expression changes are detected after application of stearate together with oleate. Furthermore, the expression of reticulocalbin-2 is significantly decreased after stearate application also in the whole cell lysate. CONCLUSIONS AND CLINICAL RELEVANCE: Several membrane-associated proteins that could be related to pro- and anti-apoptotic signaling initiated by fatty acids in human pancreatic ß-cells are identified. As far as we know, annexin A4, reticulocalbin-2, and 14-3-3γ represent novel molecules related to the effect of fatty acids on ß-cell viability.

Asunto(s)

Apoptosis/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Proteínas de la Membrana/biosíntesis , Ácido Oléico/farmacología , Ácidos Esteáricos/farmacología , Línea Celular , Humanos , Células Secretoras de Insulina/citología

RESUMEN

It has been shown that saturated fatty acids (FAs) have a detrimental effect on pancreatic ß-cells function and survival, leading to apoptosis, whereas unsaturated FAs are well tolerated and are even capable of inhibiting the pro-apoptotic effect of saturated FAs. Molecular mechanisms of apoptosis induction and regulation by FAs in ß-cells remain unclear; however, mitogen-activated protein (MAP) kinase and endoplasmic reticulum (ER) stress signaling pathways may be involved. In this study, we tested how unsaturated oleic acid (OA) affects the effect of saturated stearic acid (SA) on the p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) pathways as well as the ER stress signaling pathways during apoptosis induction in the human pancreatic ß-cells NES2Y. We demonstrated that OA is able to inhibit all effects of SA. OA alone has only minimal or no effects on tested signaling in NES2Y cells. The point of OA inhibitory intervention in SA-induced apoptotic signaling thus seems to be located upstream of the discussed signaling pathways.

Asunto(s)

Apoptosis , Estrés del Retículo Endoplásmico , Células Secretoras de Insulina/metabolismo , Sistema de Señalización de MAP Quinasas , Ácido Oléico/metabolismo , Ácidos Esteáricos/metabolismo , Línea Celular , Humanos , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/patología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo

RESUMEN

Pericellular oxygen concentration represents an important factor in the regulation of cell functions, including cell differentiation, growth and mitochondrial energy metabolism. Hypoxia in adipose tissue has been associated with altered adipokine secretion profile and suggested as a possible factor in the development of type 2 diabetes. In vitro experiments provide an indispensable tool in metabolic research, however, physical laws of gas diffusion make prolonged exposure of adherent cells to desired pericellular O2 concentrations questionable. The aim of this study was to investigate the direct effect of various O2 levels (1%, 4% and 20% O2) on the proteomic profile and triglyceride accumulation in 3T3-L1 differentiated preadipocytes using gas-permeable cultureware. Following differentiation of cells under desired pericellular O2 concentrations, cell lysates were subjected to two-dimensional gel electrophoresis and protein visualization using Coomassie blue staining. Spots showing differential expression under hypoxia were analyzed using matrix-assisted laser desorption/ionization mass spectrometry. All identified proteins were subjected to pathway analysis. We observed that protein expression of 26 spots was reproducibly affected by 4% and 1% O2 (17 upregulated and 9 downregulated). Pathway analysis showed that mitochondrial energy metabolism and triglyceride synthesis were significantly upregulated by hypoxia. In conclusion, this study demonstrated the direct effects of pericellular O2 levels on adipocyte energy metabolism and triglyceride synthesis, probably mediated through the reversed tricarboxylic acid cycle flux.

Asunto(s)

Adipocitos/citología , Diferenciación Celular/efectos de los fármacos , Gases/química , Lipogénesis/efectos de los fármacos , Oxígeno/farmacología , Proteómica , Células 3T3-L1 , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Animales , Recuento de Células , Células Cultivadas , Ciclo del Ácido Cítrico/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Electroforesis en Gel Bidimensional , Lípidos/biosíntesis , Ratones , Permeabilidad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Triglicéridos/metabolismo , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

Experiments using cultured primary cells or cell lines are a routine in vitro approach used across multiple biological disciplines, However, the structural and functional influences of various cultureware materials on cultured cells is not clearly understood. Surface treatments of cultureware have proven to have profound effects on cell viability and proliferation. In this study, we investigated the impact of polystyrene and fluorocarbon cultureware dishes on the proteomic profile of differentiated 3T3-L1 preadipocytes. After expansion and differentiation of cells on appropriate cultureware dishes, cell lysates were separated using two-dimensional gel electrophoresis and proteins were visualized with Coomassie blue staining. Spots with the highest differential expression between the two culture conditions were subsequently analyzed using matrix-assisted laser desorption/ionization mass spectrometry and the identified proteins were subjected to pathway analysis. We observed that 43% of all spots were differentially expressed depending on the cultureware. Pathway analysis revealed that glucose metabolism, mitochondrial structure and cell differentiation, represented by 14-3-3 protein-mediated signaling and the mitochondrial inner membrane organizing system (MINOS), were significantly affected by cultureware material. These results indicate that cultureware material can have a profound effect on key adipocyte functional pathways. These effects modifications of the cells should be reflected in the design of in vitro experiments and interpretation of their results.

Asunto(s)

Adipocitos/citología , Técnicas de Cultivo de Célula/instrumentación , Proteínas 14-3-3/metabolismo , Células 3T3-L1 , Adipocitos/metabolismo , Animales , Diferenciación Celular , Electroforesis en Gel Bidimensional , Regulación de la Expresión Génica , Interacciones Hidrofóbicas e Hidrofílicas , Ratones , Microscopía Confocal , Proteómica , Transducción de Señal , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Propiedades de Superficie

RESUMEN

Pollution of the environment represents one of less explored potential reasons for the worldwide epidemic of type 2 diabetes. One of the most prevalent organochlorine pollutants remains the pesticide DDT and its degradation product DDE. Despite some epidemiologic correlations between levels of DDT and DDE in human organism and the prevalence of diabetes, there is almost no information about the exact targets of these compounds inside pancreatic beta cells. To detect functional areas of pancreatic beta cells that could be affected by exposure to DDT and DDE, we analyzed changes in protein expression in the NES2Y human pancreatic beta cell line exposed to three sublethal concentrations (0.1 µM, 1 µM, 10 µM) of DDT and DDE for 1 month. Protein separation and identification was achieved using high-resolution 2D-electrophoresis, computer analysis and mass spectrometry. With these techniques, four proteins were found downregulated after exposure to 10 µM DDT: three cytoskeletal proteins (cytokeratin 8, cytokeratin 18 and actin) and one protein involved in glycolysis (alpha-enolase). Two proteins were downregulated after exposure to 10 µM DDE: cytokeratin 18 and heterogenous nuclear ribonucleoprotein H1 (HNRH1). These changes correlate with previously described effects of other stress conditions (e.g. exposure to palmitate, hyperglycemia, imidazoline derivative, and cytokines) on protein expression in pancreatic beta cells. We conclude that cytoskeletal proteins and their processing, glucose metabolism, and mRNA processing may represent targets affected by exposure to conditions hostile to pancreatic beta cells, including exposure to DDT and DDE.

Asunto(s)

DDT/toxicidad , Diclorodifenil Dicloroetileno/toxicidad , Contaminantes Ambientales/toxicidad , Células Secretoras de Insulina/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Proteínas del Citoesqueleto/metabolismo , Glucosa/metabolismo , Humanos , Células Secretoras de Insulina/metabolismo , ARN Mensajero/metabolismo

RESUMEN

Resistance of cancer cells to chemotherapeutic agents is one of the main causes of treatment failure. In order to detect proteins potentially involved in the mechanism of resistance to taxanes, we assessed differences in protein expression in MCF-7 breast cancer cells that are sensitive to paclitaxel and in the same cells with acquired resistance to paclitaxel (established in our lab). Proteins were separated using two-dimensional electrophoresis. Changes in their expression were determined and proteins with altered expression were identified using mass spectrometry. Changes in their expression were confirmed using western blot analysis. With these techniques, we found three proteins expressed differently in resistant MCF-7 cells, i.e., thyroid hormone-interacting protein 6 (TRIP6; upregulated to 650%), heat shock protein 27 (HSP27; downregulated to 50%) and cathepsin D (downregulated to 28%). Silencing of TRIP6 expression by specific siRNA leads to decreased number of grown resistant MCF-7 cells. In the present study we have pointed at some new directions in the studies of the mechanism of resistance to paclitaxel in breast cancer cells.

Asunto(s)

Antineoplásicos Fitogénicos/farmacología , Paclitaxel/farmacología , Proteoma/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Neoplasias de la Mama , Catepsina D/metabolismo , Resistencia a Antineoplásicos , Femenino , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico , Humanos , Proteínas con Dominio LIM/metabolismo , Células MCF-7 , Chaperonas Moleculares , Complejo de la Endopetidasa Proteasomal , Factores de Transcripción/metabolismo

RESUMEN

Insulin secretion in patients with manifested diabetes mellitus tends to disappear months to decades after the diagnosis, which is a clear sign of a gradual loss of pancreatic islet beta-cells. In our sample of 30 type 2 diabetic patients, whose disease manifested between 30 and 45 years of age, about a half have retained or even increased insulin secretion 30 years later, while the other half exhibit a much diminished or lost insulin secretion. Factors that can damage or destroy beta-cells can be divided into the following groups: Metabolic factors: hyperglycemia and glucotoxicity, lipotoxicity, hypoxia, reactive oxygen species; Pharmacological factors: antimicrobial medication pentamidine, SSRI antidepressants; Factors related to impaired insulin secretion: MODY type diabetes; Environmental toxic factors: rat poison Vacor, streptozotocin, polychlorinated and polybrominated hydrocarbons; Disorders of the exocrine pancreas: tumor infiltration, fibrous infiltration, chronic pancreatitis, cystic fibrosis; Infections, inflammation, autoimmunity, viral factors: Coxsackie viruses, H1N1 influenza, enteroviruses. We are currently working on finding other factors leading to beta-cell damage, studying their effect on apoptosis and necrosis and looking for possible protective factors to prevent this damage. We our increasing knowledge about the mechanisms of beta-cell damage and destruction we come ever closer to suggest measures for their prevention. In this review we offer a brief and simplified summary of some of the findings related to this area.Key words: pancreatic islet beta-cells of Langerhans - factors damaging or destroying beta-cells - insulin secretion.

RESUMEN

The resistance of cancer cells to chemotherapeutic drugs represents a major problem in cancer treatment. Despite all efforts, mechanisms of resistance have not yet been elucidated. To reveal proteins that could be involved in resistance to taxanes, we compared protein expression in whole cell lysates of SK-BR-3 breast cancer cells sensitive to paclitaxel and in lysates of the same line with acquired resistance to paclitaxel. The resistant SK-BR-3 cell line was established in our lab. Protein separation was achieved using high-resolution 2D-electrophoresis, computer analysis and mass spectro-metry. With these techniques we identified four proteins with different expression in resistant SK-BR-3 cells, i.e., serpin B3, serpin B4, heat shock protein 27 (all three upregulated) and cytokeratin 18 (downregulated). Observed changes were confirmed using western blot analysis. This study suggests new directions worthy of further study in the effort to reveal the mechanism of resistance to paclitaxel in breast cancer cells.

Asunto(s)

Adenocarcinoma/metabolismo , Antígenos de Neoplasias/biosíntesis , Neoplasias de la Mama/metabolismo , Resistencia a Antineoplásicos/fisiología , Queratina-18/biosíntesis , Serpinas/biosíntesis , Antineoplásicos Fitogénicos/farmacología , Western Blotting , Línea Celular Tumoral , Electroforesis en Gel Bidimensional , Femenino , Proteínas de Choque Térmico HSP27/biosíntesis , Humanos , Paclitaxel/farmacología , Proteómica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción

RESUMEN

Alpha-hexachlorocyclohexane (alpha-HCH), a part of the HCH pesticide mixture, is one of the most widespread persistent organic pollutants. Interestingly, only limited number of studies addressed the toxicity of alpha-HCH and the effects of its individual optical isomers have not been investigated in detail. In the present study we separated two alpha-HCH enantiomers by preparative HPLC and studied their activities towards androgen receptor (AR) using the MDA-kb2 cell line stably transfected with the luciferase reporter gene under the control of AR. There was no direct effect of alpha-HCH on AR but both isomers significantly suppressed the activity of AR in co-exposure with the natural ligand dihydrotestosterone in a concentration-dependent manner. One of the enantiomers appeared to be more active at lower concentration, which was also supported by the molecular modeling calculations with AR that showed a slight difference in estimated free energy of binding and inhibition constant between two enantiomers. Although studies with other pesticides demonstrated strong enantioselective differences in toxicity, the present research shows rather minor differences in modulations of AR by both alpha-HCH enantiomers. For the first time, enantioselective effects of alpha-HCH were demonstrated and the results suggest interaction with multiple regulatory events controlling the AR activity. Full elucidation of the toxicity mechanism will require further research.

Asunto(s)

Hexaclorociclohexano/química , Hexaclorociclohexano/farmacología , Receptores Androgénicos/metabolismo , Línea Celular , Cromatografía Líquida de Alta Presión , Dihidrotestosterona/metabolismo , Dihidrotestosterona/farmacología , Genes Reporteros , Hexaclorociclohexano/metabolismo , Humanos , Isomerismo , Luciferasas , Transfección

RESUMEN

Various synthetically readily accessible S-phenacyl xanthates are shown to undergo photoinitiated homolytic scission of the C-S bond in the primary step. The resultant fragments, phenacyl and xanthic acid radicals, recombine to form symmetrical 1,4-diketones and xanthogen disulfides, respectively, in high to moderate chemical yields in chemically inert solvents. They can also be efficiently trapped by a hydrogen-atom-donating solvent to give acetophenone and xanthic acid derivatives. The latter compound is in situ thermally converted to the corresponding alcohol in high chemical yields. S-Phenacyl xanthates could thus be utilized as synthetic precursors to the above-mentioned compounds or as photoremovable protecting groups for alcohols in which the xanthate moiety represents a photolabile linker. The photochemically released phenacyl radical fragments efficiently but reversibly add to the thiocarbonyl group of the parent xanthate molecule. The kinetics of this degenerative reversible addition-fragmentation transfer (RAFT)/macromolecular design via the interchange of xanthates (MADIX) mechanism was studied using laser flash photolysis (LFP) and density functional theory (DFT) calculations. The rate constants of the RAFT addition step, k(add) ∼ 7 × 10(8) M(-1) s(-1), and phenacyl radical addition to a double bond of 1,1-diphenylethylene, k(add) ∼ 10(8) M(-1) s(-1), in acetonitrile were experimentally determined by LFP. In addition, photoinitiation of the methyl methacrylate polymerization by S-phenacyl xanthate is demonstrated. The polydispersity index of the resulting poly(methyl methacrylate) was found to be ∼1.4. We conclude that S-phenacyl xanthates can serve simultaneously as photoinitiators as well as RAFT/MADIX agents in polymerization reactions.

RESUMEN

Tributyltin (TBT) is a widespread marine pollutant that influences physiological conditions of fish and other aquatic organisms. In addition to effects on reproduction, the immune system has been proposed as a possible target for TBT effects. In the present study, the effects of TBT exposure were examined on the expression of genes involved in immune system compentence in liver and head kidney of Atlantic salmon, in the presence and absence of a second-messenger activator (forskolin). Juvenile salmon were force-fed a diet containing TBT (0-solvent control, 0.1, 1, or 10 mg/kg fish) for 72 h. Consequently, fish from the control group and 10-mg/kg TBT group were exposed to the adenylate cyclase (AC) activator forskolin (200 µg/L) for 2 or 4 h. Forskolin was selected for this study because it is known to exhibit potent immune system enhancement by activating macrophages and lymphocytes. After sacrifice, liver and head kidney were sampled and transcript changes for interleukin (IL)-1ß, IL-10, transforming growth factor (TGF) ß, interferon (INF) α, INFγ, tumor necrosis factor (TNF) α, Mx3, and insulin-like growth factor (IGF)-1 were determined in both tissues by quantitative polymerase chain reaction (qPCR) using gene-specific primers. TBT, when given alone and also in combination with forskolin, decreased IL-1ß, TNFα, IFNγ, IFNα, Mx3, and IGF-1 gene expression. In contrast, IL-10 and TGFß transcripts were increased after TBT exposure alone and also in combination with forskolin. Generally, these effects were largely dependent on TBT dose and time of exposure when given in combination with forskolin. Overall, our findings suggest a possible immunomodulatory effect of TBT, possibly involving cAMP activation.

Asunto(s)

Colforsina/toxicidad , Desinfectantes/toxicidad , Sistema Inmunológico/efectos de los fármacos , Salmo salar/metabolismo , Compuestos de Trialquiltina/toxicidad , Contaminantes Químicos del Agua/toxicidad , Animales , Relación Dosis-Respuesta a Droga , Expresión Génica/efectos de los fármacos , Sistema Inmunológico/metabolismo , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Interferón-alfa/genética , Interferón-alfa/metabolismo , Interferón gamma/genética , Interferón gamma/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Riñón/efectos de los fármacos , Riñón/inmunología , Riñón/metabolismo , Hígado/efectos de los fármacos , Hígado/inmunología , Hígado/metabolismo , ARN Mensajero/metabolismo , Salmo salar/genética , Salmo salar/inmunología , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

The mechanisms by which organotin compounds produce modulations of the endocrine systems and other biological responses are not fully understood. In this study, juvenile salmon were force-fed diet containing TBT (0: solvent control, 0.1, 1 and 10mg/kg fish) for 72 h. Subsequently, fish exposed to solvent control and 10mg TBT were exposed to waterborne concentration (200 microg/l) of the adenylate cyclase (AC) stimulator, forskolin for 2 and 4h. The overall aim of the study was to explore whether TBT endocrine disruptive effects involve second messenger activation. Liver was sampled from individual fish (n=8) at the end of the exposures. The transcription patterns of peroxisome proliferator-activated receptor (PPAR) isotype and acyl-coenzyme A oxidase 1 (ACOX1), aromatase isoform, estrogen receptor-alpha (ER alpha), pregnane X receptor (PXR), CYP3A and glutathione S-transferase (GST) genes were measured by quantitative polymerase chain reaction (qPCR). Our data showed a consistent increase in PPAR alpha, PPAR beta and PPAR gamma mRNA and protein expression after TBT exposure that were inversely correlated with ACOX1 mRNA levels. Forskolin produced PPAR isotype-specific mRNA and protein effects that were modulated by TBT. ACOX1 expression was decreased (at 2h) and increased (at 4h) by forskolin and the presence of TBT potentiated these effects. TBT apparently increased mRNA and protein levels of cyp19a, compared to the solvent control, whereas cyp19b mRNA levels were unaffected by TBT treatment. Combined TBT and forskolin exposure produced respective decrease and increase of mRNA levels of cyp19a and cyp19b, compared with control. TBT decreased ER alpha mRNA at low dose (1mg/kg) and forskolin exposure alone produced a consistent decrease of ER alpha mRNA levels that were not affected by the presence of TBT. Interestingly, PXR and CYP3A mRNA levels were differentially affected, either decreased or increased, after exposure to TBT and forskolin, singly and also in combination. GST mRNA was increased by TBT exposure. Exposure to forskolin alone increased GST expression with time, and combined exposure with TBT potentiated these respective effects. Overall, the present study demonstrates multiple biological effects of TBT given singly or in combination with cAMP activator. There are no studies known to us that have evaluated the endocrine disruptive effects of TBT in the presence of a second messenger activator, and our data suggest that TBT may exert endocrine, biotransformation and lipid peroxidative effects through modulation of cAMP/PKA second messenger signaling with overt physiological consequences.

Asunto(s)

Exposición a Riesgos Ambientales , Regulación de la Expresión Génica/efectos de los fármacos , Hígado/efectos de los fármacos , Receptores Activados del Proliferador del Peroxisoma/metabolismo , Salmo salar/metabolismo , Compuestos de Trialquiltina/toxicidad , Contaminantes Químicos del Agua/toxicidad , Acil-CoA Oxidasa/metabolismo , Animales , Aromatasa/metabolismo , Biotransformación/efectos de los fármacos , Citocromo P-450 CYP3A/metabolismo , Receptor alfa de Estrógeno/metabolismo , Hígado/enzimología , Receptor X de Pregnano , ARN Mensajero/metabolismo , Receptores de Esteroides/metabolismo , Sistemas de Mensajero Secundario

RESUMEN

There are uncertainties regarding the role of sex steroids in sexual development and reproduction of gastropods, leading to the recent doubts as to whether organotin compounds do inhibit steroidogenic enzymes in these species. These doubts have led us to suspect that organotin compounds may affect other target molecules, particularly signal transduction molecules or secondary mediators of steroid hormone and lipid synthesis/metabolism. Therefore, we have studied the effects of TBT exposure through food on acute steroidogenesis, PPARs and CYP3A responses in the presence and absence of a cyclic AMP (cAMP) activator, forskolin. Two experiments were performed. Firstly, juvenile salmon were force-fed once with diet containing TBT doses (0.1, 1 and 10mg/kg fish) dissolved in ethanol and sampled after 72h. Secondly, fish exposed to solvent control and 10mg/kg TBT for 72h were transferred to new tanks and exposed to waterborne forskolin (200microg/L) for 2 and 4h. Our data show that juvenile salmon force-fed TBT showed modulations of multiple biological responses in interrenal tissues that include, steroidogenesis (cAMP/PKA activities; StAR and P450scc mRNA, and plasma cortisol), and mRNA for peroxisome proliferator-activated receptor (PPAR) isoforms (alpha, beta, gamma), acyl-CoA oxidase-1 (ACOX1) and CYP3A/PXR (pregnan X receptor). In addition, forskolin produced differential effects on these responses both singly and also in combination with TBT. Overall, combined forskolin and TBT exposure produced higher effects compared with TBT exposure alone, for most of the responses (cortisol, PPARbeta, ACOX1 and CYP3A). Interestingly, forskolin produced PPAR isoform-specific effects when given singly or in combination with TBT. Several TBT mediated toxicity in fish that includes thymus reduction, decrease in numbers of lymphocytes, inhibition of gonad development and masculinization, including the imposex phenomenon have been reported. When these effects are considered with the present findings, it suggests that studies on mechanisms of action or field studies may reveal endocrine, reproductive or other effects of TBT at lower concentrations than those reported to date from subchronic tests of fishes. Since the metabolic fate of organotin compounds may contribute to the toxicity of these chemicals, the present findings may represent some new aspects of TBT toxicity not previously reported.

Asunto(s)

Colforsina/toxicidad , Citocromo P-450 CYP3A/metabolismo , Receptores Activados del Proliferador del Peroxisoma/metabolismo , Receptores de Esteroides/metabolismo , Salmo salar/metabolismo , Esteroides/biosíntesis , Compuestos de Trialquiltina/toxicidad , Acil-CoA Oxidasa , Animales , Secuencia de Bases , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Citocromo P-450 CYP3A/genética , Relación Dosis-Respuesta a Droga , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/fisiología , Hidrocortisona/sangre , Hidrocortisona/metabolismo , Riñón/efectos de los fármacos , Riñón/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Receptores Activados del Proliferador del Peroxisoma/genética , Receptor X de Pregnano , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Esteroides/genética , Salmo salar/genética , Sistemas de Mensajero Secundario/efectos de los fármacos , Sistemas de Mensajero Secundario/fisiología , Factores de Tiempo , Compuestos de Trialquiltina/administración & dosificación , Contaminantes Químicos del Agua/toxicidad

RESUMEN

The mechanisms by which TBT produces modulations of the endocrine systems are not fully described. In this study, juvenile salmon were force-fed diet containing TBT (0: solvent control, 0.1, 1 and 10 mg/kg fish) for 72 h. Subsequently, fish exposed to solvent control and 10 mg/kg TBT were exposed to waterborne concentration of the adenyl cyclase stimulator forskolin (200 µg/L) for 2 and 4 h. Tissue and blood were sampled from individual fish (n=6). Gene expression patterns of CYP11ß, steroidogenic factor-1 (SF-1), and glucocorticoid receptor (GlucR) were determined by qPCR. TBT generally decreased mRNA levels of CYP11ß, GlucR and SF-1, compared to the solvent control and these effects were differentially modulated by the presence of forskolin. This study suggests that TBT may exert broader endocrine disrupting effects through possible modulation of cAMP/PKA second messenger systems.

Asunto(s)

Colforsina/farmacología , Salmón/fisiología , Compuestos de Trialquiltina/toxicidad , Contaminantes Químicos del Agua/toxicidad , Animales , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Sistema Enzimático del Citocromo P-450/efectos de los fármacos , Sistema Enzimático del Citocromo P-450/metabolismo , Disruptores Endocrinos/toxicidad , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Receptores de Glucocorticoides/efectos de los fármacos , Receptores de Glucocorticoides/metabolismo , Sistemas de Mensajero Secundario , Factor Esteroidogénico 1/efectos de los fármacos , Factor Esteroidogénico 1/metabolismo