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Trx system and the Oxidative branch of pentose phosphate pathway as targets of Thimerosal and Ethylmercury
S318
Abstracts / Toxicology Letters 238S (2015) S56–S383
P13-127 Trx system and the Oxidative branch of pentose phosphate pathway as targets of Thimerosal and Ethylmercury J. Rodrigues 1 , V. Branco 1 , J. Lu 2 , A. Holmgren 2 , C. Carvalho 1,∗ 1 2
Research Institute for Medicines (iMed.ULisboa), Lisboa, Portugal Karolinska Institutet, MBB, Stockholm, Sweden
Mercury (Hg) is a strong toxicant responsible by neurotoxicity, nefrotoxicity, cardiotoxicity and immunotoxicity besides being a teratogen. Thimerosal is still in use in medical practice as a topical antiseptic and preservative in vaccines routinely given to children while other forms of mercury, such as methylmercury represent an environmental hazard. The aim of the present study was to determine the effects of thimerosal and its metabolite ethyl mercury on the thioredoxin system and the NADP+ -dependent dehydrogenases of the pentose phosphate pathway in vitro. Results show that treatment of neuroblastoma and liver cells with thimerosal and directly with ethyl mercury decreased cell viability (IC50 : 1.5–20 M) and caused a significant decrease in the overall activities of thioredoxin, thioredoxin reductase, glucose-6-phosphate dehydrogenase and 6-phosphogulconate dehydrogenase in a dose and time-dependent manner, as a consequence, the antioxidant and protective cycle is inhibited as well as the production of NADPH would be compromised. Compared to control, the activities of Trx and TrxR in neuroblastoma cells after EtHg incubation were reduced up to 60% and 80% respectively, whereas in hepatoma cells the reduction was almost 100%. In addition, the activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were also significantly inhibited by all mercurials, with inhibition intensity of Hg2+ > MeHg ≈ EtHg > TM (p < 0.05). Cell incubation with sodium selenite partially prevented the inhibitory effects on TrxR and glucose-6-phosphate dehydrogenase. Overall, the molecular mechanism of toxicity of thimerosal and its toxic metabolite ethylmercury is related to the inhibition of the thioredoxin system and the oxidative branch of the pentose phosphate pathway. http://dx.doi.org/10.1016/j.toxlet.2015.08.906
P13-128 Oxidative stress and DNA damages induced by 1-nitropyrene in human lung fibroblasts: New insights into the mechanisms of genotoxicity and EPR-spin trapping direct monitoring of free radicals at subcellular levels M. Cassien 1,∗ , V. Tassistro 2 , M. Culcasi 1 , E. Ricquebourg 1 , S. Thétiot-Laurent 1 , A. Mercier 1 , T. Orsière 2 , S. Pietri 1 1 Aix Marseille Université, CNRS UMR 7273, SMBSO-Institut de Chimie Radicalaire, Marseille, France 2 Aix Marseille Université, CNRS UMR 7263, IMBE, Marseille, France
Polycyclic aromatic hydrocarbons (PAHs) and nitro-PAHs are ubiquitous contaminants released in the atmosphere from the incomplete combustion of organic matter and pesticide intermediates nitration. Many studies have established that 1-nitropyrene (1-NP), one the most abundant nitro-PAH pollutant in diesel exhaust, is associated with increased risk of cancer and inflammatory diseases via possible metabolic pathways, including ring oxidation, nitroreduction or conjugation reactions. In addition, genotoxicity tests (e.g. comet assay and Ames test), demonstrated
that 1-NP is a potent mutagen and genotoxic agent. However, the mechanisms underlying the promotion of DNA damages remain to be fully elucidated in relation with the progression of oxidative stress due to the stimulation of cellular free radical sources. Methods: On human lung fibroblasts exposed to environmentally relevant doses of 1-NP (1-10 microM) for 24–48 h, we performed: (i) cytokinesis-blocked micronucleus (CBMN) assay in association with immunofluorescence staining of centromere protein A in micronuclei to distinguish between induction of structural or numerical chromosome changes; (ii) EPR experiments using a set of spin trapping agents (i.e. the phosphorylated nitrone DEPMPO (Culcasi et al., 2012), the new stereoselective superoxide probe Me4 CyDEPMPO (Gosset et al., 2011)) and mitochondrial-targeted nitrones; and (iii) the evaluation of oxidative stress from protein carbonyls, lipid peroxidation and antioxidant depletion. Major results: One advantage of the CBMN assay is its ability to elucidate the genotoxic mechanism of agents with clastogenic or aneugenic modes of action. The data provide the first evidence for a superoxide-related aneugenic mode of action at low 1-NP doses and a dominant clastogenic mechanism as the 1-NP doses and exposure time increased. This two-steps mechanism differs from that induced by CeO2 -nanoparticles, an oxidant present in diesel exhausts (Benameur et al., 2014). From the EPR detection of superoxide and hydroxyl radical spin adducts, two free radical cellular sources activated by 1-NP exposure were hypothetized, i.e. NADPH oxidase and mitochondria, this latter being likely involved in oxidative stress damages development. References Culcasi, et al., 2012. Chem. Biol. Interact.. Gosset, et al., 2011. Bioorg. Med. Chem.. Benameur, et al., 2014. Nanotoxicology.
http://dx.doi.org/10.1016/j.toxlet.2015.08.907
P13-129 Impaired gut dopaminergic function in an experimental Parkinson rat model A.L. Silva 1,∗ , S. Viana 2,1 , I. Pita 1 , C. Lemos 3 , C.A. Fontes Ribeiro 2,1 , R. Prediger 3,4 , F.C. Pereira 2,1 , S. Santos 2,1 1 Laboratory of Pharmacology and Experimental Therapeutics/Institute for Biomedical Imaging and Life Sciences (IBILI), University of Coimbra, Portugal, Coimbra, Portugal 2 CNC.IBILI, University of Coimbra, Portugal, Coimbra, Portugal 3 CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Portugal, Coimbra, Portugal 4 Departamento de Farmacologia, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, UFSC, Florianópolis, Brasil, Florianópolis, Portugal
We have thoroughly demonstrated that rodents treated intranasally with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) exhibit time-dependent impairments in olfactory, emotional, cognitive and motor functions associated with disruption of dopaminergic neurotransmission across brain structures which possibly recapitulate those observed during different stages of Parkinson’s disease (PD). Although autonomic dysfunction, such as constipation, has potential sensitivity as a clinical biomarker of the premotor phase, it has been overlooked. The aim of this study was to evaluate the intestinal function before motor impairment, using this intranasal MPTP rat PD model. To this end, ileum segments from male Wistar rats (21 weeks old) were collected 7 days following the MPTP administration for functional studies.
Abstracts / Toxicology Letters 238S (2015) S56–S383
P13-127 Trx system and the Oxidative branch of pentose phosphate pathway as targets of Thimerosal and Ethylmercury J. Rodrigues 1 , V. Branco 1 , J. Lu 2 , A. Holmgren 2 , C. Carvalho 1,∗ 1 2
Research Institute for Medicines (iMed.ULisboa), Lisboa, Portugal Karolinska Institutet, MBB, Stockholm, Sweden
Mercury (Hg) is a strong toxicant responsible by neurotoxicity, nefrotoxicity, cardiotoxicity and immunotoxicity besides being a teratogen. Thimerosal is still in use in medical practice as a topical antiseptic and preservative in vaccines routinely given to children while other forms of mercury, such as methylmercury represent an environmental hazard. The aim of the present study was to determine the effects of thimerosal and its metabolite ethyl mercury on the thioredoxin system and the NADP+ -dependent dehydrogenases of the pentose phosphate pathway in vitro. Results show that treatment of neuroblastoma and liver cells with thimerosal and directly with ethyl mercury decreased cell viability (IC50 : 1.5–20 M) and caused a significant decrease in the overall activities of thioredoxin, thioredoxin reductase, glucose-6-phosphate dehydrogenase and 6-phosphogulconate dehydrogenase in a dose and time-dependent manner, as a consequence, the antioxidant and protective cycle is inhibited as well as the production of NADPH would be compromised. Compared to control, the activities of Trx and TrxR in neuroblastoma cells after EtHg incubation were reduced up to 60% and 80% respectively, whereas in hepatoma cells the reduction was almost 100%. In addition, the activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were also significantly inhibited by all mercurials, with inhibition intensity of Hg2+ > MeHg ≈ EtHg > TM (p < 0.05). Cell incubation with sodium selenite partially prevented the inhibitory effects on TrxR and glucose-6-phosphate dehydrogenase. Overall, the molecular mechanism of toxicity of thimerosal and its toxic metabolite ethylmercury is related to the inhibition of the thioredoxin system and the oxidative branch of the pentose phosphate pathway. http://dx.doi.org/10.1016/j.toxlet.2015.08.906
P13-128 Oxidative stress and DNA damages induced by 1-nitropyrene in human lung fibroblasts: New insights into the mechanisms of genotoxicity and EPR-spin trapping direct monitoring of free radicals at subcellular levels M. Cassien 1,∗ , V. Tassistro 2 , M. Culcasi 1 , E. Ricquebourg 1 , S. Thétiot-Laurent 1 , A. Mercier 1 , T. Orsière 2 , S. Pietri 1 1 Aix Marseille Université, CNRS UMR 7273, SMBSO-Institut de Chimie Radicalaire, Marseille, France 2 Aix Marseille Université, CNRS UMR 7263, IMBE, Marseille, France
Polycyclic aromatic hydrocarbons (PAHs) and nitro-PAHs are ubiquitous contaminants released in the atmosphere from the incomplete combustion of organic matter and pesticide intermediates nitration. Many studies have established that 1-nitropyrene (1-NP), one the most abundant nitro-PAH pollutant in diesel exhaust, is associated with increased risk of cancer and inflammatory diseases via possible metabolic pathways, including ring oxidation, nitroreduction or conjugation reactions. In addition, genotoxicity tests (e.g. comet assay and Ames test), demonstrated
that 1-NP is a potent mutagen and genotoxic agent. However, the mechanisms underlying the promotion of DNA damages remain to be fully elucidated in relation with the progression of oxidative stress due to the stimulation of cellular free radical sources. Methods: On human lung fibroblasts exposed to environmentally relevant doses of 1-NP (1-10 microM) for 24–48 h, we performed: (i) cytokinesis-blocked micronucleus (CBMN) assay in association with immunofluorescence staining of centromere protein A in micronuclei to distinguish between induction of structural or numerical chromosome changes; (ii) EPR experiments using a set of spin trapping agents (i.e. the phosphorylated nitrone DEPMPO (Culcasi et al., 2012), the new stereoselective superoxide probe Me4 CyDEPMPO (Gosset et al., 2011)) and mitochondrial-targeted nitrones; and (iii) the evaluation of oxidative stress from protein carbonyls, lipid peroxidation and antioxidant depletion. Major results: One advantage of the CBMN assay is its ability to elucidate the genotoxic mechanism of agents with clastogenic or aneugenic modes of action. The data provide the first evidence for a superoxide-related aneugenic mode of action at low 1-NP doses and a dominant clastogenic mechanism as the 1-NP doses and exposure time increased. This two-steps mechanism differs from that induced by CeO2 -nanoparticles, an oxidant present in diesel exhausts (Benameur et al., 2014). From the EPR detection of superoxide and hydroxyl radical spin adducts, two free radical cellular sources activated by 1-NP exposure were hypothetized, i.e. NADPH oxidase and mitochondria, this latter being likely involved in oxidative stress damages development. References Culcasi, et al., 2012. Chem. Biol. Interact.. Gosset, et al., 2011. Bioorg. Med. Chem.. Benameur, et al., 2014. Nanotoxicology.
http://dx.doi.org/10.1016/j.toxlet.2015.08.907
P13-129 Impaired gut dopaminergic function in an experimental Parkinson rat model A.L. Silva 1,∗ , S. Viana 2,1 , I. Pita 1 , C. Lemos 3 , C.A. Fontes Ribeiro 2,1 , R. Prediger 3,4 , F.C. Pereira 2,1 , S. Santos 2,1 1 Laboratory of Pharmacology and Experimental Therapeutics/Institute for Biomedical Imaging and Life Sciences (IBILI), University of Coimbra, Portugal, Coimbra, Portugal 2 CNC.IBILI, University of Coimbra, Portugal, Coimbra, Portugal 3 CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Portugal, Coimbra, Portugal 4 Departamento de Farmacologia, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, UFSC, Florianópolis, Brasil, Florianópolis, Portugal
We have thoroughly demonstrated that rodents treated intranasally with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) exhibit time-dependent impairments in olfactory, emotional, cognitive and motor functions associated with disruption of dopaminergic neurotransmission across brain structures which possibly recapitulate those observed during different stages of Parkinson’s disease (PD). Although autonomic dysfunction, such as constipation, has potential sensitivity as a clinical biomarker of the premotor phase, it has been overlooked. The aim of this study was to evaluate the intestinal function before motor impairment, using this intranasal MPTP rat PD model. To this end, ileum segments from male Wistar rats (21 weeks old) were collected 7 days following the MPTP administration for functional studies.