RESUMO
Polycyclic aromatic hydrocarbons (PAHs) are lipophilic compounds able to accumulate in the food chain. Mussels showed to bioaccumulate contaminants, such as PAHs, so that recurrent consumption of such contaminated food represents a risk for human health. This study was aimed to elucidate if acute exposure of Mediterranean blue mussel (Mytilus galloprovincialis), a bivalve of great economic importance in several countries, to a PAH, benzo[a]pyrene (B[a]P), at doses able to induce cytochrome P450 1A (CYP1A) and pathological changes in mussel gills, can produce accumulation in soft tissue. We explored the cytotoxic effects (cell viability, DNA laddering, and glutathione levels) of in vitro exposure of human peripheral blood mononuclear cells (PBMCs) to organic extracts obtained from blue mussels previously exposed for 12 and 72h via water to B[a]P (0.5-1mg/L). In our experimental conditions, B[a]P induced CYP1A induction and morphological changes in mussel gills and a significant B[a]P accumulation in soft tissue. Conversely, exposing PBMCs to organic extracts obtained from contaminated mussels, resulted in a significant reduction of cell viability and cell glutathione content, and in an increase in DNA laddering. This confirms that consumption of mussels from B[a]P polluted waters might affect human health. Our data lead us to suggest that CYP1A activity in mussel gills may be useful (more than the amount of detected PAHs in the mussel edible tissue) as a marker in assessment of risk for health of consumers exposed to PAHs through ingestion of shellfish.
Assuntos
Benzo(a)pireno/toxicidade , Mytilus edulis/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Benzo(a)pireno/análise , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Brânquias/química , Brânquias/efeitos dos fármacos , Brânquias/patologia , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Medição de Risco , Poluentes Químicos da Água/análiseRESUMO
AIMS/HYPOTHESIS: The pathogenic role of excessive vascular endothelial growth factor (VEGF)-A in diabetic nephropathy has not been defined. We sought to test whether increased podocyte VEGF-A signalling determines the severity of diabetic glomerulopathy. METHODS: Podocyte-specific, doxycycline-inducible Vegf164 (the most abundant Vegfa isoform) overexpressing adult transgenic mice were made diabetic with low doses of streptozotocin and examined 12 weeks after onset of diabetes. We studied diabetic and non-diabetic transgenic mice fed a standard or doxycycline-containing diet. VEGF-A and albuminuria were measured by ELISA, creatinine was measured by HPLC, renal morphology was examined by light and electron microscopy, and gene expression was assessed by quantitative PCR, immunoblotting and immunohistochemistry. RESULTS: Podocyte Vegf164 overexpression in our mouse model of diabetes resulted in advanced diabetic glomerulopathy, characterised by Kimmelstiel-Wilson-like nodular glomerulosclerosis, microaneurysms, mesangiolysis, glomerular basement membrane thickening, podocyte effacement and massive proteinuria associated with hyperfiltration. It also led to increased VEGF receptor 2 and semaphorin3a levels, as well as nephrin and matrix metalloproteinase-2 downregulation, whereas circulating VEGF-A levels were similar to those in control diabetic mice. CONCLUSIONS/INTERPRETATION: Collectively, these data demonstrate that increased podocyte Vegf164 signalling dramatically worsens diabetic nephropathy in a streptozotocin-induced mouse model of diabetes, resulting in nodular glomerulosclerosis and massive proteinuria. This suggests that local rather than systemic VEGF-A levels determine the severity of diabetic nephropathy and that semaphorin3a signalling and matrix metalloproteinase-2 dysregulation are mechanistically involved in severe diabetic glomerulopathy.
Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/metabolismo , Podócitos/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Western Blotting , Cromatografia Líquida de Alta Pressão , Creatinina/sangue , Creatinina/urina , Diabetes Mellitus Tipo 1/genética , Nefropatias Diabéticas/genética , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase , Semaforina-3A/genética , Semaforina-3A/metabolismo , Espectrometria de Massas em Tandem , Fator A de Crescimento do Endotélio Vascular/genéticaAssuntos
Anisakis/imunologia , Asma/etiologia , Culinária , Doenças Profissionais/etiologia , Exposição Ocupacional , Animais , Asma/imunologia , Dermatite Ocupacional/etiologia , Dermatite Ocupacional/imunologia , Peixes/parasitologia , Humanos , Masculino , Doenças Profissionais/imunologia , Urticária/etiologia , Urticária/imunologia , Adulto JovemRESUMO
Chronic renal failure (CRF) is accompanied by adaptive changes in renal and extrarrenal epithelial ionic transport. Fluid reabsorption in the thick ascending limb of Henle is increased and the capacity to lower the urine osmolality in water diuresis is preserved. To study the cellular mechanism of this adaptation, we measured intracellular cAMP in microdissected medullary thick ascending limb (mTAL) segments in rats with CRF. mTAL exhibited in CRF nephrons an increase of basal cAMP from 6.0 +/- 1.5 in controls to 47.0 +/- 10.3 fmol. mm-1 tubule in CRF (P < 0.05). Maximally stimulated cAMP levels (10(-3) M IBMX plus 10(-5) M Forskolin) were different from basal levels in controls (6.0 +/- 1.5 vs 63.1 +/- 18.8, P < 0.05) but not from basal levels in CRF (47.0 +/- 10.3 vs 63.0 +/- 16.0, P = N.S.). Preincubation with the adenylate cyclase inhibitor 2'5'-dideoxyadenosine (DDA) 10(-4) M produced no changes in cAMP in controls (93.7 +/- 10.3% of DDA untreated samples) whereas it decreased to 76.2 +/- 8.8% (24% inhibition) in CRF (P < 0.05). No differences between controls and CRF groups were found in basal and stimulated cAMP in red blood cells and distal colon. The data would suggest that the cAMP pathway is an intracellular signal for mTAL adaptation in epithelial transport and that the adenylate-cyclase system is specifically activated in CRF.
Assuntos
AMP Cíclico/fisiologia , Falência Renal Crônica/fisiopatologia , Alça do Néfron/citologia , Animais , AMP Cíclico/sangue , Ativação Enzimática , Transporte de Íons , Masculino , Radioimunoensaio , Ratos , Ratos WistarRESUMO
Chronic renal failure (CRF) is accompanied by adaptive changes in electrolyte reabsorption in the thick ascending limb of Henle of surviving nephrons. To study the cellular mechanism of this adaptation, we measured intracellular cAMP in micro-dissected medullary thick ascending limb (mTAL) segments in rats with CRF. mTAL exhibited in CRF an increase of basal cAMP from 25.6 +/- 10.0 in controls to 65.8 +/- 11.3 fmol mm-1 tubule in CRF (P < 0.05). Vasopressin and calcitonin stimulated mTAL adenylate-cyclase in a dose-dependent manner in controls but failed to stimulate in CRF. Likewise, maximal stimulation with 10(-3) M 3-isobutyl-1-methylxanthine (IBMX) plus 10(-5) M forskolin increased cAMP in controls to 63.0 +/- 16.0 but not in CRF, where maximal stimulated values remained at 63.1 +/- 18.8 fmol mm-1 tubule (P NS). Alpha2-adrenoreceptor activation with clonidine at concentrations ranging from 10(-8) to 10(-6) M diminished cAMP production by 37% in CRF (P < 0.05), whereas no differences were found in controls. Thus, the basal intracellular cAMP is increased in rat mTAL in CRF. The finding that neither forskolin nor vasopressin were able to further augment intracellular cAMP would suggest that stimulatory pathways of the adenylate-cyclase system are activated in the basal state. However, mTAL cells in CRF seem to retain the response of normal epithelium to inhibitory pathways such as the one mediated by alpha2-adrenoreceptors.
Assuntos
AMP Cíclico/metabolismo , Falência Renal Crônica/metabolismo , Alça do Néfron/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Agonistas alfa-Adrenérgicos/farmacologia , Animais , Calcitonina/farmacologia , Separação Celular , Clonidina/farmacologia , Colforsina/farmacologia , Creatinina/sangue , AMP Cíclico/antagonistas & inibidores , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Taxa de Filtração Glomerular/fisiologia , Técnicas In Vitro , Masculino , Concentração Osmolar , Ratos , Ratos Wistar , Vasopressinas/farmacologiaRESUMO
Chronic renal failure (CRF) is accompanied by adaptive changes in renal and extrarrenal epithelial ionic transport. Fluid reabsorption in the thick ascending limb of Henle is increased and the capacity to lower the urine osmolality in water diuresis is preserved. To study the cellular mechanism of this adaptation, we measured intracellular cAMP in microdissected medullary thick ascending limb (mTAL) segments in rats with CRF. mTAL exhibited in CRF nephrons an increase of basal cAMP from 6.0 +/- 1.5 in controls to 47.0 +/- 10.3 fmol. mm-1 tubule in CRF (P < 0.05). Maximally stimulated cAMP levels (10(-3) M IBMX plus 10(-5) M Forskolin) were different from basal levels in controls (6.0 +/- 1.5 vs 63.1 +/- 18.8, P < 0.05) but not from basal levels in CRF (47.0 +/- 10.3 vs 63.0 +/- 16.0, P = N.S.). Preincubation with the adenylate cyclase inhibitor 25-dideoxyadenosine (DDA) 10(-4) M produced no changes in cAMP in controls (93.7 +/- 10.3
of DDA untreated samples) whereas it decreased to 76.2 +/- 8.8
(24
inhibition) in CRF (P < 0.05). No differences between controls and CRF groups were found in basal and stimulated cAMP in red blood cells and distal colon. The data would suggest that the cAMP pathway is an intracellular signal for mTAL adaptation in epithelial transport and that the adenylate-cyclase system is specifically activated in CRF.