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Alkylating agents activate chemosensing Transient Receptor Potential A1 cation channels
Abstracts / Toxicology Letters 258S (2016) S62–S324
using standard homogenization, digestion and dialysis methods using RecoverEase kits from Agilent. Background mutant frequencies (MF) from five studies were consistent with other somatic tissues. Statistically significant increases in cII MF were observed in lung (57.4 ± 15.0 × 10−2 in controls vs 154 ± 24.0 × 10−2 in ENU treatment groups) and nasal samples (54.4 ± 10.8 × 10−2 vs 416 ± 54.0 × 10−2 ). In comparison, similar background MF and increases were seen with liver (4.2-fold), glandular stomach (18.6fold) and bone marrow (14.5-fold). These results extend the range of the BB TGR mouse mutation assay to upper respiratory tract tissues thereby demonstrating robustness of the assay and the assay’s suitable for use with inhalation exposure. http://dx.doi.org/10.1016/j.toxlet.2016.06.1869 P16-031 Alkylating agents activate chemosensing Transient Receptor Potential A1 cation channels B. Stenger 1,∗ , D. Steinritz 2 , A. Tsoutsoulopoulos 2 , A. Breit 1 , H. Thiermann 2 , A. Schmidt 2 , H. John 2 , T. Popp 2 , T. Gudermann 1 1
Walther-Straub-Institute of Pharmacology and Toxicology, Ludwig-Maximilian-Universität München, Germany 2 Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany Alkylating agents have been used for chemical warfare since World War I. Recent reports from Syria and Iraq on the use of sulfur mustard (SM) by militant groups underline the acute threat by these compounds. Exposure to SM results in painful injuries affecting the respiratory system, eyes and skin. Despite decades of medical research, there is neither a specific therapy nor an antidote currently available. Toxicity provoked by highly reactive compounds has been considered as an unspecific process resulting in uncontrollable cell damage. However, recent research identified Transient Receptor Potential A1 channels (TRPA1) as cellular chemosensors for a plethora of noxious substances implicating a substantial role in molecular toxicology. TRPA1 is expressed in different human tissues including skin, lung and neuronal tissue. Here we demonstrate that SM and the monofunctional alkylating agent CEES (model substance for SMpromoted effects) are both able to activate TRPA1 channels, that results in the elevation of intracellular calcium concentrations. The TRP-channel blocker AP18 was able to prevent the CEES/SMinduced calcium response. In HEK293 cells overexpressing TRPA1, CEES cytotoxity was significantly enhanced compared to wildtype cells. TRPA1 blockers attenuated cytotoxicity, especially in the low-dose range of CEES. Similar results were found in A549 lung epithelial cells, endogenously expressing TRPA1. These cells also responded after CEES/SM challenge with a distinct calcium influx that could be diminished by AP18. In summary, TRPA1 channel are involved in the pathophysiology of alkylating compounds and drugs addressing these channels may represent a promising approach to mitigate alkylating agent induced cell damage. http://dx.doi.org/10.1016/j.toxlet.2016.06.1870
S245
P16-032 CeO2 nanoparticle attenuates paraquat-induced liver injury by decreasing oxidative stress and DNA damage A. Ranjbar ∗ , H. Heidary Dartoti, S. Soleimani Asl, F. Firozian, M. Taheri Azandariani Cerium oxide nanoparticle (CeNPs) is one of the most widely used and most important nanoparticles in addition to having strong antioxidative properties and inhibiting free radicals. Paraquat (PQ), an effective and widely used herbicide, has been proven to be safe when appropriately applied to eliminate weeds. The purpose of this study was to investigate the effect of CeNPs against PQ-induced liver injury in association with its antioxidant activity. The male rats were treated intraperitoneally daily with PQ (50 mg/kg/day) and CeNPs (15, 30 and 60 mg/kg/day) were administered alone or in combination for 14 days. After treatments, serum and liver tissue samples were collected from all rats. Oxidative stress biomarkers include total antioxidant capacity (TAC), lipid peroxidation (LPO), total thiol groups (TTG), DNA damage and NO levels were carried out. The results have shown that PQ induces oxidation stress and hepatic tissue damage in comparison to control group, CeNPs especially in 15 mg/kg dose has antioxidative effect and taking this combination with PQ compromises the damage. Oxidative stress and consequently hepatic damage induced by PQ can be reduced by CeNPs in lower doses. http://dx.doi.org/10.1016/j.toxlet.2016.06.1871 P16-033 Molecular mechanisms of alkylphenol-mediated endocrine disruption in Leydig cells P. Labohá 1,∗ , T. Jambor 2 , A. Yawer 1 , N. Lukáˇc 2 , I. Sovadinová 1 1
Research Centre for Toxic Compounds in the Environment (Recetox), Faculty of Science, Masaryk University, Brno, Czech Republic 2 Department of Animal Physiology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture, Nitra, Slovak Republic Recently, a dramatic decline of male reproduction function has been reported, associated with impairment of testicular functions and fertility. Nonylphenol (NP) and octylphenol (OP) are widespread man-made alkylphenolic environmental contaminants with endocrine disruptive properties listed among the EU priority hazardous substances. NP and OP have been shown to decrease sperm fertilizing ability and functions of male reproductive system. Both these alkylphenols interfere with estrogen and androgen receptors, and thus with proper functioning of natural hormones that are essential for normal development of male reproductive system. However, there is still a limited knowledge on different molecular mechanisms underlying their adverse effects on male reproduction system. Our study focused on epigenetic mechanisms that could be involved in NP and OP toxicity, namely on gap junctional intercellular communication (GJIC) through connexin (Cx) channels. Cx43-based GJIC between Leydig cells plays crucial roles in proper testicular steroidogenesis and hormonal control of spermatogenesis. We determined the effects of NP and OP on viability and proliferation of mouse Leydig TM3 cells (ATCC) and assessed their ability to disrupt GJIC in TM3 cells and alter Cx43 expression and phosphorylation. Since TM3 cells express estrogen receptor (ER), we used specific inhibitors of different signalling pathways to determine if the effects on GJIC are mediated via ER or via different
using standard homogenization, digestion and dialysis methods using RecoverEase kits from Agilent. Background mutant frequencies (MF) from five studies were consistent with other somatic tissues. Statistically significant increases in cII MF were observed in lung (57.4 ± 15.0 × 10−2 in controls vs 154 ± 24.0 × 10−2 in ENU treatment groups) and nasal samples (54.4 ± 10.8 × 10−2 vs 416 ± 54.0 × 10−2 ). In comparison, similar background MF and increases were seen with liver (4.2-fold), glandular stomach (18.6fold) and bone marrow (14.5-fold). These results extend the range of the BB TGR mouse mutation assay to upper respiratory tract tissues thereby demonstrating robustness of the assay and the assay’s suitable for use with inhalation exposure. http://dx.doi.org/10.1016/j.toxlet.2016.06.1869 P16-031 Alkylating agents activate chemosensing Transient Receptor Potential A1 cation channels B. Stenger 1,∗ , D. Steinritz 2 , A. Tsoutsoulopoulos 2 , A. Breit 1 , H. Thiermann 2 , A. Schmidt 2 , H. John 2 , T. Popp 2 , T. Gudermann 1 1
Walther-Straub-Institute of Pharmacology and Toxicology, Ludwig-Maximilian-Universität München, Germany 2 Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany Alkylating agents have been used for chemical warfare since World War I. Recent reports from Syria and Iraq on the use of sulfur mustard (SM) by militant groups underline the acute threat by these compounds. Exposure to SM results in painful injuries affecting the respiratory system, eyes and skin. Despite decades of medical research, there is neither a specific therapy nor an antidote currently available. Toxicity provoked by highly reactive compounds has been considered as an unspecific process resulting in uncontrollable cell damage. However, recent research identified Transient Receptor Potential A1 channels (TRPA1) as cellular chemosensors for a plethora of noxious substances implicating a substantial role in molecular toxicology. TRPA1 is expressed in different human tissues including skin, lung and neuronal tissue. Here we demonstrate that SM and the monofunctional alkylating agent CEES (model substance for SMpromoted effects) are both able to activate TRPA1 channels, that results in the elevation of intracellular calcium concentrations. The TRP-channel blocker AP18 was able to prevent the CEES/SMinduced calcium response. In HEK293 cells overexpressing TRPA1, CEES cytotoxity was significantly enhanced compared to wildtype cells. TRPA1 blockers attenuated cytotoxicity, especially in the low-dose range of CEES. Similar results were found in A549 lung epithelial cells, endogenously expressing TRPA1. These cells also responded after CEES/SM challenge with a distinct calcium influx that could be diminished by AP18. In summary, TRPA1 channel are involved in the pathophysiology of alkylating compounds and drugs addressing these channels may represent a promising approach to mitigate alkylating agent induced cell damage. http://dx.doi.org/10.1016/j.toxlet.2016.06.1870
S245
P16-032 CeO2 nanoparticle attenuates paraquat-induced liver injury by decreasing oxidative stress and DNA damage A. Ranjbar ∗ , H. Heidary Dartoti, S. Soleimani Asl, F. Firozian, M. Taheri Azandariani Cerium oxide nanoparticle (CeNPs) is one of the most widely used and most important nanoparticles in addition to having strong antioxidative properties and inhibiting free radicals. Paraquat (PQ), an effective and widely used herbicide, has been proven to be safe when appropriately applied to eliminate weeds. The purpose of this study was to investigate the effect of CeNPs against PQ-induced liver injury in association with its antioxidant activity. The male rats were treated intraperitoneally daily with PQ (50 mg/kg/day) and CeNPs (15, 30 and 60 mg/kg/day) were administered alone or in combination for 14 days. After treatments, serum and liver tissue samples were collected from all rats. Oxidative stress biomarkers include total antioxidant capacity (TAC), lipid peroxidation (LPO), total thiol groups (TTG), DNA damage and NO levels were carried out. The results have shown that PQ induces oxidation stress and hepatic tissue damage in comparison to control group, CeNPs especially in 15 mg/kg dose has antioxidative effect and taking this combination with PQ compromises the damage. Oxidative stress and consequently hepatic damage induced by PQ can be reduced by CeNPs in lower doses. http://dx.doi.org/10.1016/j.toxlet.2016.06.1871 P16-033 Molecular mechanisms of alkylphenol-mediated endocrine disruption in Leydig cells P. Labohá 1,∗ , T. Jambor 2 , A. Yawer 1 , N. Lukáˇc 2 , I. Sovadinová 1 1
Research Centre for Toxic Compounds in the Environment (Recetox), Faculty of Science, Masaryk University, Brno, Czech Republic 2 Department of Animal Physiology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture, Nitra, Slovak Republic Recently, a dramatic decline of male reproduction function has been reported, associated with impairment of testicular functions and fertility. Nonylphenol (NP) and octylphenol (OP) are widespread man-made alkylphenolic environmental contaminants with endocrine disruptive properties listed among the EU priority hazardous substances. NP and OP have been shown to decrease sperm fertilizing ability and functions of male reproductive system. Both these alkylphenols interfere with estrogen and androgen receptors, and thus with proper functioning of natural hormones that are essential for normal development of male reproductive system. However, there is still a limited knowledge on different molecular mechanisms underlying their adverse effects on male reproduction system. Our study focused on epigenetic mechanisms that could be involved in NP and OP toxicity, namely on gap junctional intercellular communication (GJIC) through connexin (Cx) channels. Cx43-based GJIC between Leydig cells plays crucial roles in proper testicular steroidogenesis and hormonal control of spermatogenesis. We determined the effects of NP and OP on viability and proliferation of mouse Leydig TM3 cells (ATCC) and assessed their ability to disrupt GJIC in TM3 cells and alter Cx43 expression and phosphorylation. Since TM3 cells express estrogen receptor (ER), we used specific inhibitors of different signalling pathways to determine if the effects on GJIC are mediated via ER or via different