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Structure–activity studies on the RXR agonist activity of organotins
Abstracts / Toxicology Letters 221S (2013) S59–S256
drastically contributes towards total anti-oxidant activity. Approximately 10-11 times higher TPx was observed for valine derived diselenide with non bonding nitrogen interactions. The compound also displayed significantly (p < 0.05) higher activity in DPPH radical scavenging and deoxyribose degradation assays. Using biological tissues i.e. rat’s brain preparation, the compound protected against free radical production as measured by 2,7-dichlorofluorescin diacetate (DCFH-DA) assay and inhibited TBARS production as measured by malondialdehyde (MDA) levels. When the non bonding interactions were blocked i.e. protected nitrogen with butoxycarbonyl (BOC) group, the resultant activity drastically decreased, evident from both computational and wet experiments. The toxicological profile of the most active compound indicated that in-vivo treatment with valine derived selenide is not associated with increased lipid and protein oxidation, free radical production, inhibition of thiol containing enzymes, vitamin C levels and renal (urea and creatinine) and hepatic (ALT and AST) toxicity. This is the 1st comprehensive report which describes the influence of nonbonding interaction of this interesting class of organochalcogens. http://dx.doi.org/10.1016/j.toxlet.2013.05.379
P13-23 Structure–activity studies on the RXR agonist activity of organotins Tsuyoshi Nakanishi 1,∗ , Youhei Hiromori 2 , Jun-ichi Nishikawa 3 , Hisamitsu Nagase 1 Gifu Pharmaceutical University, Gifu, Japan, 2 Kinjo Gakuin University, Nagoya, Japan, 3 Mukogawa Women’s University, Nishinomiya, Japan
1
We previously reported that tributyltin (TBT) and triphenyltin (TPT) are potential endocrine disruptors to function as retinoid X receptor (RXR) agonist. To extend our knowledge of the correlation between the structures of organotins and their RXR agonist activity, we here assessed RXR agonist activity of various organotin compounds using human RXR␣ (hRXR␣) mutants with altered ligand binding potential. The most compounds active in wild hRXR␣ were TBT and TPT derivatives. Furthermore, tripropyltin, tripentyltin and tricyclohexyltin were also active. Unlike the above-active compounds, the dealkylated- and dearylated-triorganotin compounds had no effect on hRXR␣ transactivation. The results suggest that the trialkyl or triaryl substituents are required for organotins to act as RXR agonists. However, the structure of triorganotins is entirely distinct from established RXR ligands such as 9-cis retinoic acid and rexinoids. Previous X-ray crystallographic studies have revealed that an acidic head group and a long aliphatic/aromatic chain in rexinoids interact with R316 in helix 5 and some subsets of binding pocket residues in helix 11, respectively. By contrast, TBT interacts with only a subset of binding pocket residues including C432 in helix 11. Thereupon, we investigated critical amino acid residues of hRXR␣ for transactivation induced by the active triorganotins to mutate each residue to alanine. We found that all tested active triorganotins also activated the R316A mutant hRXR␣ but failed to activate C432A mutant hRXR␣. Our results suggest that C432 is crtical for transactivation by triorganotins but R316 are not required, and triorganotins and retinoids have distinct ligand-binding properties of RXR. http://dx.doi.org/10.1016/j.toxlet.2013.05.380
S173
P13-24 Tributyltin-induced endoplasmic reticulum stress and its mechanism Yaichiro Kotake ∗ , Midori Isomura, Kyoichi Masuda, Masatsugu Miyara, Katsuhiro Okuda, Shigeyoshi Samizo, Seigo Sanoh, Toru Hosoi, koichiro Ozawa, Shigeru Ohta Hiroshima University, Hiroshima, Japan Organotin compounds, especially tributyltin chloride (TBT), have been widely used in antifouling paints for marine vessels, but exhibit various toxicities in mammals. The endoplasmic reticulum (ER) is a multifunctional organelle that controls posttranslational modification and intracellular Ca2+ signaling. When the capacity of the quality control system of ER is exceeded under stress including ER Ca2+ homeostasis disruption, ER functions are impaired and unfolded proteins are accumulated in ER lumen, which is called ER stress. Here, we examined whether TBT causes ER stress in human neuroblastoma SH-SY5Y cells. We found that TBT induced ER stress markers such as CHOP, GRP78, spliced XBP1 mRNA and phosphorylated eIF2␣. TBT also decreased the cell viability both concentration- and time-dependently. Dibutyltin and monobutyltin did not induce ER stress markers. We hypothesized that TBT induces ER stress via Ca2+ depletion, and to test this idea, we examined the effect of TBT on intracellular Ca2+ concentration using fura-2 AM, a Ca2+ fluorescent probe. TBT increased intracellular Ca2+ concentration in a TBT-concentration-dependent manner, and this increase was mainly blocked by dantrolene (a ryanodine receptor inhibitor). Dantrolene also partially but significantly inhibited TBT-induced GRP78 expression and cell death. These results suggest that TBT increases intracellular Ca2+ concentration by releasing Ca2+ from ER, thereby causing ER stress. http://dx.doi.org/10.1016/j.toxlet.2013.05.381
P13-25 Zinc supplementation protects from cadmium-induced oxidative stress in the brain of rats Malgorzata M. Brzóska ∗ , Joanna Rogalska, Malgorzata Galazyn-Sidorczuk Department of Toxicology, Medical University of Bialystok, Poland Oxidative stress is one of the mechanisms of toxic action of cadmium (Cd). This metal may indirectly generate formation of reactive oxygen species in various organs and tissues leading to their oxidative damage. The brain tissue, due to high presence of mitochondria and high oxygen turnover as well as high content of polyunsaturated fatty acids and iron, is especially vulnerable to development of oxidative stress. The aim of this study was to investigate whether zinc (Zn), characterized by antioxidative properties, may protect against cadmium-induced oxidative stress and its consequences in the brain. For this purpose, enzymatic (glutathione peroxidase, superoxide dismutase, catalase) and non-enzymatic (glutathione) antioxidants, total antioxidative and oxidative status, hydrogen peroxide, and markers of oxidative lipid (F2 -isoprostanes and lipid peroxides) and protein (protein carbonyl groups) damage as well as cadmium and zinc were determined in the brain tissue of the male Wistar rats administered 30 mg Zn/l or/and 5 mg Cd/l for 6 months. Moreover, index of oxidative stress was calculated.
drastically contributes towards total anti-oxidant activity. Approximately 10-11 times higher TPx was observed for valine derived diselenide with non bonding nitrogen interactions. The compound also displayed significantly (p < 0.05) higher activity in DPPH radical scavenging and deoxyribose degradation assays. Using biological tissues i.e. rat’s brain preparation, the compound protected against free radical production as measured by 2,7-dichlorofluorescin diacetate (DCFH-DA) assay and inhibited TBARS production as measured by malondialdehyde (MDA) levels. When the non bonding interactions were blocked i.e. protected nitrogen with butoxycarbonyl (BOC) group, the resultant activity drastically decreased, evident from both computational and wet experiments. The toxicological profile of the most active compound indicated that in-vivo treatment with valine derived selenide is not associated with increased lipid and protein oxidation, free radical production, inhibition of thiol containing enzymes, vitamin C levels and renal (urea and creatinine) and hepatic (ALT and AST) toxicity. This is the 1st comprehensive report which describes the influence of nonbonding interaction of this interesting class of organochalcogens. http://dx.doi.org/10.1016/j.toxlet.2013.05.379
P13-23 Structure–activity studies on the RXR agonist activity of organotins Tsuyoshi Nakanishi 1,∗ , Youhei Hiromori 2 , Jun-ichi Nishikawa 3 , Hisamitsu Nagase 1 Gifu Pharmaceutical University, Gifu, Japan, 2 Kinjo Gakuin University, Nagoya, Japan, 3 Mukogawa Women’s University, Nishinomiya, Japan
1
We previously reported that tributyltin (TBT) and triphenyltin (TPT) are potential endocrine disruptors to function as retinoid X receptor (RXR) agonist. To extend our knowledge of the correlation between the structures of organotins and their RXR agonist activity, we here assessed RXR agonist activity of various organotin compounds using human RXR␣ (hRXR␣) mutants with altered ligand binding potential. The most compounds active in wild hRXR␣ were TBT and TPT derivatives. Furthermore, tripropyltin, tripentyltin and tricyclohexyltin were also active. Unlike the above-active compounds, the dealkylated- and dearylated-triorganotin compounds had no effect on hRXR␣ transactivation. The results suggest that the trialkyl or triaryl substituents are required for organotins to act as RXR agonists. However, the structure of triorganotins is entirely distinct from established RXR ligands such as 9-cis retinoic acid and rexinoids. Previous X-ray crystallographic studies have revealed that an acidic head group and a long aliphatic/aromatic chain in rexinoids interact with R316 in helix 5 and some subsets of binding pocket residues in helix 11, respectively. By contrast, TBT interacts with only a subset of binding pocket residues including C432 in helix 11. Thereupon, we investigated critical amino acid residues of hRXR␣ for transactivation induced by the active triorganotins to mutate each residue to alanine. We found that all tested active triorganotins also activated the R316A mutant hRXR␣ but failed to activate C432A mutant hRXR␣. Our results suggest that C432 is crtical for transactivation by triorganotins but R316 are not required, and triorganotins and retinoids have distinct ligand-binding properties of RXR. http://dx.doi.org/10.1016/j.toxlet.2013.05.380
S173
P13-24 Tributyltin-induced endoplasmic reticulum stress and its mechanism Yaichiro Kotake ∗ , Midori Isomura, Kyoichi Masuda, Masatsugu Miyara, Katsuhiro Okuda, Shigeyoshi Samizo, Seigo Sanoh, Toru Hosoi, koichiro Ozawa, Shigeru Ohta Hiroshima University, Hiroshima, Japan Organotin compounds, especially tributyltin chloride (TBT), have been widely used in antifouling paints for marine vessels, but exhibit various toxicities in mammals. The endoplasmic reticulum (ER) is a multifunctional organelle that controls posttranslational modification and intracellular Ca2+ signaling. When the capacity of the quality control system of ER is exceeded under stress including ER Ca2+ homeostasis disruption, ER functions are impaired and unfolded proteins are accumulated in ER lumen, which is called ER stress. Here, we examined whether TBT causes ER stress in human neuroblastoma SH-SY5Y cells. We found that TBT induced ER stress markers such as CHOP, GRP78, spliced XBP1 mRNA and phosphorylated eIF2␣. TBT also decreased the cell viability both concentration- and time-dependently. Dibutyltin and monobutyltin did not induce ER stress markers. We hypothesized that TBT induces ER stress via Ca2+ depletion, and to test this idea, we examined the effect of TBT on intracellular Ca2+ concentration using fura-2 AM, a Ca2+ fluorescent probe. TBT increased intracellular Ca2+ concentration in a TBT-concentration-dependent manner, and this increase was mainly blocked by dantrolene (a ryanodine receptor inhibitor). Dantrolene also partially but significantly inhibited TBT-induced GRP78 expression and cell death. These results suggest that TBT increases intracellular Ca2+ concentration by releasing Ca2+ from ER, thereby causing ER stress. http://dx.doi.org/10.1016/j.toxlet.2013.05.381
P13-25 Zinc supplementation protects from cadmium-induced oxidative stress in the brain of rats Malgorzata M. Brzóska ∗ , Joanna Rogalska, Malgorzata Galazyn-Sidorczuk Department of Toxicology, Medical University of Bialystok, Poland Oxidative stress is one of the mechanisms of toxic action of cadmium (Cd). This metal may indirectly generate formation of reactive oxygen species in various organs and tissues leading to their oxidative damage. The brain tissue, due to high presence of mitochondria and high oxygen turnover as well as high content of polyunsaturated fatty acids and iron, is especially vulnerable to development of oxidative stress. The aim of this study was to investigate whether zinc (Zn), characterized by antioxidative properties, may protect against cadmium-induced oxidative stress and its consequences in the brain. For this purpose, enzymatic (glutathione peroxidase, superoxide dismutase, catalase) and non-enzymatic (glutathione) antioxidants, total antioxidative and oxidative status, hydrogen peroxide, and markers of oxidative lipid (F2 -isoprostanes and lipid peroxides) and protein (protein carbonyl groups) damage as well as cadmium and zinc were determined in the brain tissue of the male Wistar rats administered 30 mg Zn/l or/and 5 mg Cd/l for 6 months. Moreover, index of oxidative stress was calculated.