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Effect of platycodon grandiflorum root-derived saponins on nitric oxide synthase in human endothelial cells
Abstracts / Toxicology Letters 196S (2010) S37–S351
tein metabolism, is primarily excreted across the gill membranes. When the excretion of ammonia is impaired, the serum ammonia concentration increases. In the present study, an effect of elevated serum ammonia concentration on histopathology of gills and other tissues of common carp (Cyprinus carpio L.) was investigated. The fish were subjected to: (1) high water pH (10), (2) increased ammonia concentration at high water pH (10), (3) high water pH (10) and simultaneously per os administration of ammonium salt (NH4 Cl) mixed into the amyloidal vehicle, and (4) combination of exposure and per os administration of ammonia. In all the treatment groups, significant elevation of serum ammonia was observed after 24 h compared to control, which was kept in ammonia-free water at pH of 6.3 and per os administrated by ammonia-free amyloidal vehicle. The serum ammonia concentrations were as follows: control – 282 ± 21 mol l−1 , group 1 – 948 ± 66 mol l−1 , group 2 – 1098 ± 73 mol l−1 , group 3 – 2965 ± 149 mol l−1 , group 4 – 4466 ± 233 mol l−1 N-ammonia. All the treatments caused histopathological changes in gills of common carp similar to toxic necrosis, but the severity of these changes was positively correlated with the ammonia concentration in the blood serum. Therefore, the results showed that elevated ammonia concentrations in blood serum and toxic necrosis of carp gills as well can also be caused by high pH of water whereas the effect of pH can be even higher when the fish are fed with diet containing N-substances. Acknowledgements. This study was supported by the grant nos. MSM6007665809, QH82117 and MSM6215712402. doi:10.1016/j.toxlet.2010.03.786
P208-021 Biochemical and histological evaluations of dicarboximide fungicide vinclozolin on pregnant Wistar albino rats E.A. Koc¸kaya 1 , E. Karacao˘glu 2 , A. Kılıc¸ 2 , G. Selmano˘glu 2 1
Gazi University, Turkey, 2 Hacettepe University, Turkey
Vinclozolin is a dicarboximide fungicide which is used for protecting fruits and vegetables against various fungal pathogens. This fungicide is used on food crops such as grapes, ornamentals and also turf grass. It has been identified as an anti-androgenic endocrine distrupter that is related to androgen inhibition in rats. It has two metabolites, M1 and M2 and these metabolites display anti-andorgenic properties. Human can expose to this fungicide via digesting the vinclozolin-treated vegetables and fruits. In this study, 2.5–3 weeks aged Wistar albino female rats were mated to Wistar albino male rats. Vaginal smear samples were evaluated for gestation. The beginning of gestation was confirmed by the presence of spermatozoa in vaginal smear samples on the following morning, and it is determined as gestation day 0 (GD 0). Pregnant rats were divided into 4 groups, 8 rats in each group. Vinclozolin was administered at 50 mg/kg/day and 100 mg/kg/day orally by dissolving corn oil for 20 days. Rats were dissected on the day 20 of gestation and blood samples were taken for whole blood count and biochemical analysis such as AST, ALT, ALP, urea and creatinine. Additionally, liver and kidney of rats were dissected out for morphological and histopathological analysis. As a consequence of this study, there were no changes on the whole blood count and biochemical parameters. And also there were no changes on the weights of liver and kidney. However, some histopathological changes were observed. doi:10.1016/j.toxlet.2010.03.787
S235
P208-022 Melatonin limits cyclosporine toxicity by reducing ER chaperones in the rat A. Stacchiotti, F. Bonomini, C. Rossini, R. Rezzani Department of Biomedical Sciences and Biotechnology, University of Brescia, Italy Cyclosporine (CsA) has been widely used to prevent allograft rejection in transplantation and to treat autoimmune diseases even if it exerts severe side-effects in the kidney, but also in the heart and liver. Melatonin (Mel), the pineal hormone, has been reported to be effective against CsA-induced cell-death, fibrosis, hypertension and oxidative/nitrosactive damage in target organs. The endoplasmic reticulum (ER) is essential for the maturation of cell surface and secretory proteins and maintenance of Ca2+ homeostasis. ER-alteration leads to unfolded protein response (UPR) that upregulates ER chaperones (GRP78 and GRP94) to counteract cell death and Ca2+ dyshomeostasis. Aims of this in vivo study were: (1) to test the role of ER stress/UPR analysing GRP78 and GRP94 expressions in rat kidney, heart and liver after CsA schedule for 30 days and (2) to link beneficial effect of melatonin to decrease or inhibition of ER stress/UPR and related pro-apoptotic marker such as CHOP. Sprague–Dawley rats were divided into four groups which were treated with olive oil (CsA vehicle), Mel alone, CsA and CsA plus Mel. Kidney, heart and liver were treated for morphological analysis and immunohistochemistry of ER chaperones and CHOP. The results indicate that in CsA group, GRP78 is overexpressed in the renal cortex and in pericentral and parenchymal liver zones; in contrast after CsA plus Mel, GRP78 expression was limited. CHOP was detected in the nuclei of tubular epithelial cells in the kidney and greatly in hepatocytes after CsA treatment but disappeared after combined Mel. Using CsA schedule, GRP94 staining was evident in the heart, in the distal tubules in the outer medulla and moderate in periportal area in the liver. In Mel plus CsA group GRP94 was undetectable. We suggest that the beneficial role of Mel against CsA-toxicity might be directly or indirectly associated to ER stress modulation. doi:10.1016/j.toxlet.2010.03.788
P208-023 Effect of platycodon grandiflorum root-derived saponins on nitric oxide synthase in human endothelial cells H.G. Kim, H.G. Jeong Chungnam National University, Republic of Korea Nitric oxide (NO) produced by endothelial nitric-oxide synthase (eNOS) represents an antithrombotic and antiatherosclerotic principle in the vasculature. Previous study, we demonstrated that the saponins derived from roots of Platycodon grandiflorum (CKS) inhibited tumor necrosis factor-alpha induced expression of adhesion molecules in human endothelial cells. In this study, we identified that CKS increased of eNOS phosphorylation and NO production in human endothelial cells. Treatment of CKS increases the phosphorylation of Akt, p38/MAPK, AMP-activated protein kinase (AMPK) and calmodulin-dependent protein kinase II (CaM kinase II) leading to increase NO production in cells. Furthermore, Inhibitors of Akt (LY294002), p38/MAPK (SB203580), AMPK (compound C) and CaM kinase II (W7) failed to suppress the CKS induced eNOS phosphorylation. In addition, CKS induced eNOS phosphorylation was inhibited by dominant negative mutant form of AMPK overex-
S236
Abstracts / Toxicology Letters 196S (2010) S37–S351
pression (DN-AMPK). Taken together, these results indicated that CKS stimulates eNOS phosphorylation and NO production via activation of PI3K/Akt, p38/MAPK, AMPK and CaM kinase II. doi:10.1016/j.toxlet.2010.03.789
P208-024 Combination effects of PCB126 and 17beta-estradiol in human endothelial cells H. Andersson, E. Brittebo Uppsala University, Sweden Epidemiological and experimental evidence suggests that exposure to dioxin-like polychlorinated biphenyls (PCBs) is a risk factor for cardiovascular disease. The effects of dioxin-like PCBs are largely mediated by ligand-dependent activation of the aryl hydrocarbon receptor (AhR) followed by up-regulation of cytochrome P450 (CYP) 1 enzymes. Other mechanisms such as increased proinflammatory cell signalling and oxidative stress may, however, also contribute to the harmful effects. Humans are constantly exposed to estrogenic compounds through pharmaceuticals and industrial chemicals. It is previously known that estrogen can modulate the effects of AhR ligands but little is known about combination effects of estrogen and AhR ligands on the endothelium. The aim of this study was to study the combination effects of the dioxin-like PCB126 (1 M) and physiological levels (10 nM) of 17beta-estradiol (E2) in human umbilical vein endothelial cells (HUVEC). PCB126s effects on markers of endothelial dysfunction and CYP1 enzymes were examined in the presence and absence of E2. The preliminary results suggest that 24 h simultaneous treatment with E2 and PCB126 significantly increased COX-2 gene and protein expression in HUVEC compared to PCB126 alone or vehicle. Also, the PCB126-induced CYP1A1 and CYP1B1 gene expression was potentiated in the presence of E2. The enhanced effects on COX-2 and CYP1 expression following combination treatment was not observed after 1 h incubation time. The results also showed a reduced production of nitric oxide (NO) and increased production of reactive oxygen species (ROS) in HUVEC following 1 and 8 h treatment with PCB126, respectively. The PCB126-induced effects on NO and ROS did not change in the presence of E2 but was inhibited by simultaneous treatment with an AhR antagonist. These preliminary results suggest that physiological levels of E2 may, in a time-dependent manner, enhance some of PCB126s effects on human endothelial cells. doi:10.1016/j.toxlet.2010.03.790
P208-025 A study on histopathological changes of gastric parietal cells observed in beagle dogs with decreased food consumption O. Sawamoto, T. Fukuda, Y. Hayami, Y. Nakashima Otsuka Pharmaceutical Factory, Inc., Japan Although noise is the predominant source of work-related hearing loss, evidence has demonstrated that chemical toxicants can also cause hearing loss and enhance sensitivity to noise. The adverse auditory effects of chemical toxicants have been investigated more systematically during the past two decades, both in animal and human field and clinical studies. Existing evidence has prompted the proposal of new guidelines and standards on hearing loss prevention. In the U.S., the National Institute for Occupational Safety
and Health has discussed specific research needs regarding the ototoxicity of chemicals used at work. The American Conference of Governmental Industrial Hygienists and the U.S. Army have proposed preliminary practical steps that employers and occupational health professionals can take to improve hearing loss prevention. Australia and New Zealand have developed standard AS/NZS 1269:2005, recommending hearing tests for workers exposed to ototoxic agents. In the legislative arena, the European Parliament published a new noise directive (2003/10/EC), to be adopted by all participants’ countries. This Directive requires employers to give attention to any effects on workers’ health and safety resulting from interactions between noise and work-related ototoxic substances, when performing risk assessment of workplaces. Legislation regarding compensation has also changed in Australia (Workcover Guides for the Evaluation of Hearing Impaired, June 2002) and Brazil (Decree 3048, May 6, 1999). A new concept of creating an ototoxicity notation has been proposed. This presentation will examine the recent guidelines and legislative developments and discuss alternative strategies for preventing auditory effects of exposure to ototoxic chemicals. Disclaimer: The findings and conclusions in this abstract have not been formally disseminated by the National Institute for Occupational Safety and Health and should not be construed to represent any agency determination or policy. doi:10.1016/j.toxlet.2010.03.791
P208-026 ECG acquisition in freely-moving cynomolgus monkeys using external telemetry for toxicology (ET2) system versus conventional (snapshot) ECG recording in chair-restrained animals. Sensitivity validation with dofetilide, a QT-interval prolonging drug J.P. Briffaux, E. Chalencon, C. Bory, P. Legé, S. Baudet, S. Milano MDS Pharma Services, Lyon, France The stress associated with restraint of cynomolgus monkeys, known for their highly labile cardiovascular parameters, may hamper the reliability of snapshot ECGs collected during toxicology studies. In addition, the short duration of ECGs collected by this approach precludes performing qualitative analysis of ECG waveforms and rhythm. Finally, the restraint of individual animals in chairs renders ECG collection time- and resource consuming. Bluetooth technology allows the simultaneous recording of surface ECG data collected by external telemetry in a large number of freemoving monkeys. Surface ECGs were recorded in six free-moving cynomolgus monkeys using an external telemetry system for toxicology (ET2), based on the JET (DSI) technology. The monkeys were equipped with a JET device housed in al jacket one day before the start of recording. Four days after the ET2 session, snapshot (paper based) ECGs were collected from the same six animals placed in a restraining chair., A 6 lead ECG trace was recorded during both sessions. The heart rate and duration of the RR, PR, QT, QTc intervals, and of the QRS complex were analysed with Ponemah-P3 Plus (DSI) for ET2collected ECGs and by manual reading with a calliper from paper ECGs. In both sessions, the animals were orally administered vehicle or dofetilide at doses of 0.05 and 0.2 mg/kg (according to a Latin square design). ECGs were recorded from 0.5 h before, to 24 h after dosing and subsequently analysed at pre-defined time-points. Both systems detected the dofetilide-induced QT interval prolongation but its time- and dose dependency were visible only with ET2.
tein metabolism, is primarily excreted across the gill membranes. When the excretion of ammonia is impaired, the serum ammonia concentration increases. In the present study, an effect of elevated serum ammonia concentration on histopathology of gills and other tissues of common carp (Cyprinus carpio L.) was investigated. The fish were subjected to: (1) high water pH (10), (2) increased ammonia concentration at high water pH (10), (3) high water pH (10) and simultaneously per os administration of ammonium salt (NH4 Cl) mixed into the amyloidal vehicle, and (4) combination of exposure and per os administration of ammonia. In all the treatment groups, significant elevation of serum ammonia was observed after 24 h compared to control, which was kept in ammonia-free water at pH of 6.3 and per os administrated by ammonia-free amyloidal vehicle. The serum ammonia concentrations were as follows: control – 282 ± 21 mol l−1 , group 1 – 948 ± 66 mol l−1 , group 2 – 1098 ± 73 mol l−1 , group 3 – 2965 ± 149 mol l−1 , group 4 – 4466 ± 233 mol l−1 N-ammonia. All the treatments caused histopathological changes in gills of common carp similar to toxic necrosis, but the severity of these changes was positively correlated with the ammonia concentration in the blood serum. Therefore, the results showed that elevated ammonia concentrations in blood serum and toxic necrosis of carp gills as well can also be caused by high pH of water whereas the effect of pH can be even higher when the fish are fed with diet containing N-substances. Acknowledgements. This study was supported by the grant nos. MSM6007665809, QH82117 and MSM6215712402. doi:10.1016/j.toxlet.2010.03.786
P208-021 Biochemical and histological evaluations of dicarboximide fungicide vinclozolin on pregnant Wistar albino rats E.A. Koc¸kaya 1 , E. Karacao˘glu 2 , A. Kılıc¸ 2 , G. Selmano˘glu 2 1
Gazi University, Turkey, 2 Hacettepe University, Turkey
Vinclozolin is a dicarboximide fungicide which is used for protecting fruits and vegetables against various fungal pathogens. This fungicide is used on food crops such as grapes, ornamentals and also turf grass. It has been identified as an anti-androgenic endocrine distrupter that is related to androgen inhibition in rats. It has two metabolites, M1 and M2 and these metabolites display anti-andorgenic properties. Human can expose to this fungicide via digesting the vinclozolin-treated vegetables and fruits. In this study, 2.5–3 weeks aged Wistar albino female rats were mated to Wistar albino male rats. Vaginal smear samples were evaluated for gestation. The beginning of gestation was confirmed by the presence of spermatozoa in vaginal smear samples on the following morning, and it is determined as gestation day 0 (GD 0). Pregnant rats were divided into 4 groups, 8 rats in each group. Vinclozolin was administered at 50 mg/kg/day and 100 mg/kg/day orally by dissolving corn oil for 20 days. Rats were dissected on the day 20 of gestation and blood samples were taken for whole blood count and biochemical analysis such as AST, ALT, ALP, urea and creatinine. Additionally, liver and kidney of rats were dissected out for morphological and histopathological analysis. As a consequence of this study, there were no changes on the whole blood count and biochemical parameters. And also there were no changes on the weights of liver and kidney. However, some histopathological changes were observed. doi:10.1016/j.toxlet.2010.03.787
S235
P208-022 Melatonin limits cyclosporine toxicity by reducing ER chaperones in the rat A. Stacchiotti, F. Bonomini, C. Rossini, R. Rezzani Department of Biomedical Sciences and Biotechnology, University of Brescia, Italy Cyclosporine (CsA) has been widely used to prevent allograft rejection in transplantation and to treat autoimmune diseases even if it exerts severe side-effects in the kidney, but also in the heart and liver. Melatonin (Mel), the pineal hormone, has been reported to be effective against CsA-induced cell-death, fibrosis, hypertension and oxidative/nitrosactive damage in target organs. The endoplasmic reticulum (ER) is essential for the maturation of cell surface and secretory proteins and maintenance of Ca2+ homeostasis. ER-alteration leads to unfolded protein response (UPR) that upregulates ER chaperones (GRP78 and GRP94) to counteract cell death and Ca2+ dyshomeostasis. Aims of this in vivo study were: (1) to test the role of ER stress/UPR analysing GRP78 and GRP94 expressions in rat kidney, heart and liver after CsA schedule for 30 days and (2) to link beneficial effect of melatonin to decrease or inhibition of ER stress/UPR and related pro-apoptotic marker such as CHOP. Sprague–Dawley rats were divided into four groups which were treated with olive oil (CsA vehicle), Mel alone, CsA and CsA plus Mel. Kidney, heart and liver were treated for morphological analysis and immunohistochemistry of ER chaperones and CHOP. The results indicate that in CsA group, GRP78 is overexpressed in the renal cortex and in pericentral and parenchymal liver zones; in contrast after CsA plus Mel, GRP78 expression was limited. CHOP was detected in the nuclei of tubular epithelial cells in the kidney and greatly in hepatocytes after CsA treatment but disappeared after combined Mel. Using CsA schedule, GRP94 staining was evident in the heart, in the distal tubules in the outer medulla and moderate in periportal area in the liver. In Mel plus CsA group GRP94 was undetectable. We suggest that the beneficial role of Mel against CsA-toxicity might be directly or indirectly associated to ER stress modulation. doi:10.1016/j.toxlet.2010.03.788
P208-023 Effect of platycodon grandiflorum root-derived saponins on nitric oxide synthase in human endothelial cells H.G. Kim, H.G. Jeong Chungnam National University, Republic of Korea Nitric oxide (NO) produced by endothelial nitric-oxide synthase (eNOS) represents an antithrombotic and antiatherosclerotic principle in the vasculature. Previous study, we demonstrated that the saponins derived from roots of Platycodon grandiflorum (CKS) inhibited tumor necrosis factor-alpha induced expression of adhesion molecules in human endothelial cells. In this study, we identified that CKS increased of eNOS phosphorylation and NO production in human endothelial cells. Treatment of CKS increases the phosphorylation of Akt, p38/MAPK, AMP-activated protein kinase (AMPK) and calmodulin-dependent protein kinase II (CaM kinase II) leading to increase NO production in cells. Furthermore, Inhibitors of Akt (LY294002), p38/MAPK (SB203580), AMPK (compound C) and CaM kinase II (W7) failed to suppress the CKS induced eNOS phosphorylation. In addition, CKS induced eNOS phosphorylation was inhibited by dominant negative mutant form of AMPK overex-
S236
Abstracts / Toxicology Letters 196S (2010) S37–S351
pression (DN-AMPK). Taken together, these results indicated that CKS stimulates eNOS phosphorylation and NO production via activation of PI3K/Akt, p38/MAPK, AMPK and CaM kinase II. doi:10.1016/j.toxlet.2010.03.789
P208-024 Combination effects of PCB126 and 17beta-estradiol in human endothelial cells H. Andersson, E. Brittebo Uppsala University, Sweden Epidemiological and experimental evidence suggests that exposure to dioxin-like polychlorinated biphenyls (PCBs) is a risk factor for cardiovascular disease. The effects of dioxin-like PCBs are largely mediated by ligand-dependent activation of the aryl hydrocarbon receptor (AhR) followed by up-regulation of cytochrome P450 (CYP) 1 enzymes. Other mechanisms such as increased proinflammatory cell signalling and oxidative stress may, however, also contribute to the harmful effects. Humans are constantly exposed to estrogenic compounds through pharmaceuticals and industrial chemicals. It is previously known that estrogen can modulate the effects of AhR ligands but little is known about combination effects of estrogen and AhR ligands on the endothelium. The aim of this study was to study the combination effects of the dioxin-like PCB126 (1 M) and physiological levels (10 nM) of 17beta-estradiol (E2) in human umbilical vein endothelial cells (HUVEC). PCB126s effects on markers of endothelial dysfunction and CYP1 enzymes were examined in the presence and absence of E2. The preliminary results suggest that 24 h simultaneous treatment with E2 and PCB126 significantly increased COX-2 gene and protein expression in HUVEC compared to PCB126 alone or vehicle. Also, the PCB126-induced CYP1A1 and CYP1B1 gene expression was potentiated in the presence of E2. The enhanced effects on COX-2 and CYP1 expression following combination treatment was not observed after 1 h incubation time. The results also showed a reduced production of nitric oxide (NO) and increased production of reactive oxygen species (ROS) in HUVEC following 1 and 8 h treatment with PCB126, respectively. The PCB126-induced effects on NO and ROS did not change in the presence of E2 but was inhibited by simultaneous treatment with an AhR antagonist. These preliminary results suggest that physiological levels of E2 may, in a time-dependent manner, enhance some of PCB126s effects on human endothelial cells. doi:10.1016/j.toxlet.2010.03.790
P208-025 A study on histopathological changes of gastric parietal cells observed in beagle dogs with decreased food consumption O. Sawamoto, T. Fukuda, Y. Hayami, Y. Nakashima Otsuka Pharmaceutical Factory, Inc., Japan Although noise is the predominant source of work-related hearing loss, evidence has demonstrated that chemical toxicants can also cause hearing loss and enhance sensitivity to noise. The adverse auditory effects of chemical toxicants have been investigated more systematically during the past two decades, both in animal and human field and clinical studies. Existing evidence has prompted the proposal of new guidelines and standards on hearing loss prevention. In the U.S., the National Institute for Occupational Safety
and Health has discussed specific research needs regarding the ototoxicity of chemicals used at work. The American Conference of Governmental Industrial Hygienists and the U.S. Army have proposed preliminary practical steps that employers and occupational health professionals can take to improve hearing loss prevention. Australia and New Zealand have developed standard AS/NZS 1269:2005, recommending hearing tests for workers exposed to ototoxic agents. In the legislative arena, the European Parliament published a new noise directive (2003/10/EC), to be adopted by all participants’ countries. This Directive requires employers to give attention to any effects on workers’ health and safety resulting from interactions between noise and work-related ototoxic substances, when performing risk assessment of workplaces. Legislation regarding compensation has also changed in Australia (Workcover Guides for the Evaluation of Hearing Impaired, June 2002) and Brazil (Decree 3048, May 6, 1999). A new concept of creating an ototoxicity notation has been proposed. This presentation will examine the recent guidelines and legislative developments and discuss alternative strategies for preventing auditory effects of exposure to ototoxic chemicals. Disclaimer: The findings and conclusions in this abstract have not been formally disseminated by the National Institute for Occupational Safety and Health and should not be construed to represent any agency determination or policy. doi:10.1016/j.toxlet.2010.03.791
P208-026 ECG acquisition in freely-moving cynomolgus monkeys using external telemetry for toxicology (ET2) system versus conventional (snapshot) ECG recording in chair-restrained animals. Sensitivity validation with dofetilide, a QT-interval prolonging drug J.P. Briffaux, E. Chalencon, C. Bory, P. Legé, S. Baudet, S. Milano MDS Pharma Services, Lyon, France The stress associated with restraint of cynomolgus monkeys, known for their highly labile cardiovascular parameters, may hamper the reliability of snapshot ECGs collected during toxicology studies. In addition, the short duration of ECGs collected by this approach precludes performing qualitative analysis of ECG waveforms and rhythm. Finally, the restraint of individual animals in chairs renders ECG collection time- and resource consuming. Bluetooth technology allows the simultaneous recording of surface ECG data collected by external telemetry in a large number of freemoving monkeys. Surface ECGs were recorded in six free-moving cynomolgus monkeys using an external telemetry system for toxicology (ET2), based on the JET (DSI) technology. The monkeys were equipped with a JET device housed in al jacket one day before the start of recording. Four days after the ET2 session, snapshot (paper based) ECGs were collected from the same six animals placed in a restraining chair., A 6 lead ECG trace was recorded during both sessions. The heart rate and duration of the RR, PR, QT, QTc intervals, and of the QRS complex were analysed with Ponemah-P3 Plus (DSI) for ET2collected ECGs and by manual reading with a calliper from paper ECGs. In both sessions, the animals were orally administered vehicle or dofetilide at doses of 0.05 and 0.2 mg/kg (according to a Latin square design). ECGs were recorded from 0.5 h before, to 24 h after dosing and subsequently analysed at pre-defined time-points. Both systems detected the dofetilide-induced QT interval prolongation but its time- and dose dependency were visible only with ET2.