Toxicological effects of thiomersal and ethylmercury: Inhibition of the thioredoxin system and NADP+-dependent dehydrogenases of the pentose phosphate pathway
“…Only when compound (EtHg and MeHg) concentrations rose to 5 μM, did the oxidation of Trx1 increase significantly. In line with the fact that EtHg is the most toxic Hg compound to SH-SY5Y cells and in agreement with previous findings [10], EtHg-mediated Trx1 oxidation was visible as soon as after 6 h of exposure. On the other hand, Hg 2+ did not produce any significant change in the oxidation state of Trx1, which could explain its lower toxicity for SH-SY5Y cells.…”
Section: Discussionsupporting
confidence: 93%
“…1), with the respective LC 50 after 24 h of exposure being 4.6, 4.9 and 40.8 μM. These results are in agreement with the general notion that organomercurials are more cytotoxic than inorganic forms and that EtHg has a slightly higher toxicity than MeHg [10].Fig. 1General cell death as assessed by LDH assay after exposure of SH-SY5Y cells to mercury compounds.…”
Section: Resultssupporting
confidence: 86%
“…The selenoenzyme TrxR is extremely prone to inhibition by mercurials both in vitro [3], [4], [10] and in vivo [11], [12]. For example, in HeLa cells exposed for 24 h to MeHg the IC 50 for TrxR was 1.4 µM whereas for the homologous enzyme, glutathione reductase (GR), it was > 20 µM [3].…”
Mercury (Hg) compounds target both cysteine (Cys) and selenocysteine (Sec) residues in peptides and proteins. Thus, the components of the two major cellular antioxidant systems – glutathione (GSH) and thioredoxin (Trx) systems – are likely targets for mercurials. Hg exposure results in GSH depletion and Trx and thioredoxin reductase (TrxR) are prime targets for mercury. These systems have a wide-range of common functions and interaction between their components has been reported. However, toxic effects over both systems are normally treated as isolated events.To study how the interaction between the glutathione and thioredoxin systems is affected by Hg, human neuroblastoma (SH-SY5Y) cells were exposed to 1 and 5 μM of inorganic mercury (Hg2+), methylmercury (MeHg) or ethylmercury (EtHg) and examined for TrxR, GSH and Grx levels and activities, as well as for Trx redox state. Phosphorylation of apoptosis signalling kinase 1 (ASK1), caspase-3 activity and the number of apoptotic cells were evaluated to investigate the induction of Trx-mediated apoptotic cell death. Additionally, primary cerebellar neurons from mice depleted of mitochondrial Grx2 (mGrx2D) were used to examine the link between Grx activity and Trx function.Results showed that Trx was affected at higher exposure levels than TrxR, especially for EtHg. GSH levels were only significantly affected by exposure to a high concentration of EtHg. Depletion of GSH with buthionine sulfoximine (BSO) severely increased Trx oxidation by Hg. Notably, EtHg-induced oxidation of Trx was significantly enhanced in primary neurons of mGrx2D mice. Our results suggest that GSH/Grx acts as backups for TrxR in neuronal cells to maintain Trx turnover during Hg exposure, thus linking different mechanisms of molecular and cellular toxicity. Finally, Trx oxidation by Hg compounds was associated to apoptotic hallmarks, including increased ASK-1 phosphorylation, caspase-3 activation and increased number of apoptotic cells.
“…Only when compound (EtHg and MeHg) concentrations rose to 5 μM, did the oxidation of Trx1 increase significantly. In line with the fact that EtHg is the most toxic Hg compound to SH-SY5Y cells and in agreement with previous findings [10], EtHg-mediated Trx1 oxidation was visible as soon as after 6 h of exposure. On the other hand, Hg 2+ did not produce any significant change in the oxidation state of Trx1, which could explain its lower toxicity for SH-SY5Y cells.…”
Section: Discussionsupporting
confidence: 93%
“…1), with the respective LC 50 after 24 h of exposure being 4.6, 4.9 and 40.8 μM. These results are in agreement with the general notion that organomercurials are more cytotoxic than inorganic forms and that EtHg has a slightly higher toxicity than MeHg [10].Fig. 1General cell death as assessed by LDH assay after exposure of SH-SY5Y cells to mercury compounds.…”
Section: Resultssupporting
confidence: 86%
“…The selenoenzyme TrxR is extremely prone to inhibition by mercurials both in vitro [3], [4], [10] and in vivo [11], [12]. For example, in HeLa cells exposed for 24 h to MeHg the IC 50 for TrxR was 1.4 µM whereas for the homologous enzyme, glutathione reductase (GR), it was > 20 µM [3].…”
Mercury (Hg) compounds target both cysteine (Cys) and selenocysteine (Sec) residues in peptides and proteins. Thus, the components of the two major cellular antioxidant systems – glutathione (GSH) and thioredoxin (Trx) systems – are likely targets for mercurials. Hg exposure results in GSH depletion and Trx and thioredoxin reductase (TrxR) are prime targets for mercury. These systems have a wide-range of common functions and interaction between their components has been reported. However, toxic effects over both systems are normally treated as isolated events.To study how the interaction between the glutathione and thioredoxin systems is affected by Hg, human neuroblastoma (SH-SY5Y) cells were exposed to 1 and 5 μM of inorganic mercury (Hg2+), methylmercury (MeHg) or ethylmercury (EtHg) and examined for TrxR, GSH and Grx levels and activities, as well as for Trx redox state. Phosphorylation of apoptosis signalling kinase 1 (ASK1), caspase-3 activity and the number of apoptotic cells were evaluated to investigate the induction of Trx-mediated apoptotic cell death. Additionally, primary cerebellar neurons from mice depleted of mitochondrial Grx2 (mGrx2D) were used to examine the link between Grx activity and Trx function.Results showed that Trx was affected at higher exposure levels than TrxR, especially for EtHg. GSH levels were only significantly affected by exposure to a high concentration of EtHg. Depletion of GSH with buthionine sulfoximine (BSO) severely increased Trx oxidation by Hg. Notably, EtHg-induced oxidation of Trx was significantly enhanced in primary neurons of mGrx2D mice. Our results suggest that GSH/Grx acts as backups for TrxR in neuronal cells to maintain Trx turnover during Hg exposure, thus linking different mechanisms of molecular and cellular toxicity. Finally, Trx oxidation by Hg compounds was associated to apoptotic hallmarks, including increased ASK-1 phosphorylation, caspase-3 activation and increased number of apoptotic cells.
“…TRX1, a physiological inhibitor of ASK1, is a 12-kDa ubiquitous protein with a redox-active disulfide/dithiol within the conserved active site sequence (-Cys-Gly-Pro-Cys-) (29). Oxidized TRX1 with a disulfide on its active site is reduced by NADPH-dependent thioredoxin reductase1 (TR1) to restore its functions (30,31). TRX1 combined with ASK1 to form a TRX1-ASK1 complex which in return inactivated the ASK1 protein expression (27).…”
Claudin 6 (CLDN6), a member of tight junction protein claudin (CLDN) family, inhibits proliferation and induces apoptosis of MCF-7 breast cancer cells. However, these molecular mechanisms of CLDN6-induced apoptosis remain largely elusive. We previously found that restoration of human CLDN6 gene expression was correlated with the expression level of apoptosis signal-regulating kinase 1 (ASK1) using cDNA array and bioinformatics analysis. ASK1, a mitogen-activated protein kinase kinase kinase, is involved in environmental stress-activation of the c-jun N-terminal kinase (JNK) and p38 pathways, which contribute to apoptosis-associated tumor cell death. In the present study, we show that the restoration of CLDN6 gene expression in MCF-7 cells marhedly decreased ASK1 phosphorylation at Ser967. Activated ASK1ser967 further induced the activation of downstream targets, JNK and p38 kinase. MCF-7/CLDN6 stable transfection cell clone treated with TRX1, an ASK1 inhibitor, showed suppressed JNK and p38 activation, and showed substantially increased survival and colony formation and reduced percent of apoptotic cells using TUNEL staining and DNA ladder. Furthermore, TRX1 treatment increased Bcl-2/Bax ratio and reduced caspase-3 cleavage in MCF-7/CLDN6 stable transfection cell clone. Therefore, these data show that CLDN6 mediates ASK1-p38/JNK apoptotic signaling in MCF-7 cells, and it is correlated with constitutive deregulation of the balance of Bcl-2 family proteins and activation of caspase-3.
“…TrxR is a prime target for mercurials due to the high reactivity and location of the Sec residue at the open Cterminal (34,35). Several in vitro and in vivo studies have shown a significant drop in TrxR activity in the brain following exposure to different Hg compounds, which is both time and concentration dependent (27,28,166,220). In liver cells, inhibition of cytosolic TrxR1 by Hg 2+ is counteracted by upregulation of TrxR1 synthesis via the Nrf2 pathway (29).…”
Further study on the roles of thiol-dependent redox systems in the CNS will improve our understanding of the pathogenesis of many human neuronal disorders and also help to develop novel protective and therapeutic strategies against neuronal diseases. Antioxid. Redox Signal. 27, 989-1010.
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