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Comparison of acetaminophen hepatotoxicity and nephrotoxicity in vitro
Abstracts / Toxicology Letters 229S (2014) S40–S252
and Ca2+ ). The thrombolytic activity was evaluated in vitro and rBP-SP induced clots lysis in a time-dependent manner. P. pastoris expression system is an efficient, rapid and cheap method for obtaining recombinant SVSPs, since yeast are capable of promoting post-translational modifications and provide an adequate folding of proteins rich in disulfide bonds. Moreover, heterologous expression of SVSPs may enable a more detailed functional characterization and the pharmacological application of these toxins. http://dx.doi.org/10.1016/j.toxlet.2014.06.307 P-1.128 Comparison of acetaminophen hepatotoxicity and nephrotoxicity in vitro Tomas Rousar 1,2,∗ , Martina Vrbova 1,3 , Otto Kucera 2 , Erika Nydlova 1,3 , Zuzana Cervinkova 2 1 Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic, 2 Department of Physiology, Faculty of Medicine in Hradec Králové, Charles University in Prague, Hradec Králové, Czech Republic, 3 Department of Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic
Acetaminophen (APAP) is a frequently used analgesic and antipyretic. The overdose can cause hepatotoxicity and nephrotoxicity. Acetaminophen toxicity has been considered as the most often cause of acute liver failure and that is why APAP hepatotoxicity has been studied in a number of in vitro studies. On the other hand, acetaminophen nephrotoxicity has been estimated only in some experiments. Therefore, the goal of our study was to compare hepatotoxic and nephrotoxic effect of acetaminophen in vitro. We used cultivated rat hepatocytes to characterize APAP hepatotoxicity. To elucidate the acetaminophen nephrotoxicity, we used human kidney cells (HK-2). Both cell types were incubated with acetaminophen (0–10 mM) for up to 24 h. We measured the LDH leakage, WST-1 to determine the changes in viability. Glutathione content and ROS production was measured for detection of oxidative stress. We found that acetaminophen induced typical glutathione depletion in 5 mM APAP treated hepatocytes after 6 h although no significant depletion was found after 24 h in HK-2 cells. After 24 h, there was no glutathione detected in hepatocytes. On the other hand, ROS production was enhanced in both types of cells significantly; e.g. in HK-2 cells, 10 mM APAP caused the increase in ROS production by 100%. We conclude, we proved that acetaminophen is able to induce toxic impairment in both tested cell types in relation to the dose and time of incubation but the extent of toxic impairment is lower in kidney cells than in hepatocytes. The study was supported by grant MZCR NT/14320-3/2013. http://dx.doi.org/10.1016/j.toxlet.2014.06.308 P-1.129 BaltMPI: A new metalloprotease inhibitor from Bothrops alternatus snake serum Tatiana Zapata Palacio 1 , Norival Alves Santos-Filho 2 , Benedito Barraviera 3 , Rui Seabra Ferreira Junior 3 , José Cesar Rosa 4 , Suely Vilela Sampaio 1,∗ 1
Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, USP, Ribeirão Preto, SP, Brazil, 2 Unidade de Síntese, Estrutura e Aplicac¸ões de Peptídeos e Proteínas, UNESP, Araraquara, SP, Brazil, 3 CEVAP, UNESP,
S81
Botucatu, SP, Brazil, 4 Centro de Química de Proteínas, Faculdade de Medicina de Ribeirão Preto, USP, Ribeirão Preto, SP, Brazil Introduction: Metalloproteases are proteolytic enzymes present in several snake venoms. These proteins have important roles in the typical physiopathology of Bothrops envenomations. It is known that inhibitory compounds are present in serum, plasma and/or muscles of venomous snakes, and they are involved in a protective mechanism avoiding possible damages caused to the snake by its own venom. The aim of this work was the isolation and biochemical characterization of a novel metalloprotease inhibitor from Bothrops alternatus snake serum. Methods: Lyophilized B. alternatus snake serum was fractionated by four consecutive chromatographic steps: a DEAE-Sepharose, a size exclusion chromatography on Superdex200, an ion exchange chromatography on MonoQ and a reverse phase chromatography. The selection of the fraction of interest was made by the hemorrhagic activity, using a metalloprotease from B. atrox snake venom (Batroxase) as a positive control. The inhibitory potential was observed by the absence of formation of hemorrhagic halos in mice in comparison to the positive control. Results and conclusion: The obtained protein showed a single band with molecular mass around 66 kDa by SDS-PAGE, and 68.4 kDa by MALDI-TOF-MS and PI 4.0. The protein was named BaltMPI, and presented homology with a metalloprotease inhibitor from B. jararaca serum. The protein presented a inhibitory dose of 5.0 mg against Batroxase’s minimal hemorrhage dose (10 mg), with effective dose (ED50 ) of 0.982 mg. The characterization of inhibitory proteins as BaltMPI is very important as it could enable their use as antagonists of the damaging effects of snake venom metalloproteases. Financial support: CNPq, CAPES; FAPESP; NAP-TOXAN-USP. http://dx.doi.org/10.1016/j.toxlet.2014.06.309 P-1.130 The role of global histone modifications in Fumonisin B1-induced toxicity Duygu Sancak ∗ , Sibel Ozden Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Istanbul University, Istanbul, Turkey Fumonisin B1 (FB1) is the most prevalent member of a family of toxins produced by several species of Fusarium moulds which occur mainly in maize. Due to structural similarities with sphingolipid, FB1 exerts its toxic effects by disruption of sphingolipid biosynthesis, accumulation of sphinganine which can be highly cytotoxic and affects a wide variety of cellular system. FB1 was related to esophageal cancer in humans and was reported to be carcinogenic in rats. The mechanisms of underlying FB1 carcinogenicity are still unclear. Therefore, non-genotoxic (epigenetic) may play an important role in the mechanism of FB1 carcinogenicity. The aim of this study was to investigate dose and time dependent effects of FB1 on global histone modifications such as H3K9 methylation and H3K9 acetylation and the enzymes involved this mechanisms in rat kidney epithelial cells (NRK-52E). The increased level of global histone H3K9 di- and tri-methylation were observed in FB1-treated cells while level of global histone H3K9 acetylation was decreased. FB1 caused some changes on the activities of histone methyltransferase and histone acetyltransferase at high concentrations in NRK-52E cells. Further, the effects of trichostatin A, a histone deacetylase inhibitor, was investigated in NRK-52E cells and caused increase on
and Ca2+ ). The thrombolytic activity was evaluated in vitro and rBP-SP induced clots lysis in a time-dependent manner. P. pastoris expression system is an efficient, rapid and cheap method for obtaining recombinant SVSPs, since yeast are capable of promoting post-translational modifications and provide an adequate folding of proteins rich in disulfide bonds. Moreover, heterologous expression of SVSPs may enable a more detailed functional characterization and the pharmacological application of these toxins. http://dx.doi.org/10.1016/j.toxlet.2014.06.307 P-1.128 Comparison of acetaminophen hepatotoxicity and nephrotoxicity in vitro Tomas Rousar 1,2,∗ , Martina Vrbova 1,3 , Otto Kucera 2 , Erika Nydlova 1,3 , Zuzana Cervinkova 2 1 Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic, 2 Department of Physiology, Faculty of Medicine in Hradec Králové, Charles University in Prague, Hradec Králové, Czech Republic, 3 Department of Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic
Acetaminophen (APAP) is a frequently used analgesic and antipyretic. The overdose can cause hepatotoxicity and nephrotoxicity. Acetaminophen toxicity has been considered as the most often cause of acute liver failure and that is why APAP hepatotoxicity has been studied in a number of in vitro studies. On the other hand, acetaminophen nephrotoxicity has been estimated only in some experiments. Therefore, the goal of our study was to compare hepatotoxic and nephrotoxic effect of acetaminophen in vitro. We used cultivated rat hepatocytes to characterize APAP hepatotoxicity. To elucidate the acetaminophen nephrotoxicity, we used human kidney cells (HK-2). Both cell types were incubated with acetaminophen (0–10 mM) for up to 24 h. We measured the LDH leakage, WST-1 to determine the changes in viability. Glutathione content and ROS production was measured for detection of oxidative stress. We found that acetaminophen induced typical glutathione depletion in 5 mM APAP treated hepatocytes after 6 h although no significant depletion was found after 24 h in HK-2 cells. After 24 h, there was no glutathione detected in hepatocytes. On the other hand, ROS production was enhanced in both types of cells significantly; e.g. in HK-2 cells, 10 mM APAP caused the increase in ROS production by 100%. We conclude, we proved that acetaminophen is able to induce toxic impairment in both tested cell types in relation to the dose and time of incubation but the extent of toxic impairment is lower in kidney cells than in hepatocytes. The study was supported by grant MZCR NT/14320-3/2013. http://dx.doi.org/10.1016/j.toxlet.2014.06.308 P-1.129 BaltMPI: A new metalloprotease inhibitor from Bothrops alternatus snake serum Tatiana Zapata Palacio 1 , Norival Alves Santos-Filho 2 , Benedito Barraviera 3 , Rui Seabra Ferreira Junior 3 , José Cesar Rosa 4 , Suely Vilela Sampaio 1,∗ 1
Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, USP, Ribeirão Preto, SP, Brazil, 2 Unidade de Síntese, Estrutura e Aplicac¸ões de Peptídeos e Proteínas, UNESP, Araraquara, SP, Brazil, 3 CEVAP, UNESP,
S81
Botucatu, SP, Brazil, 4 Centro de Química de Proteínas, Faculdade de Medicina de Ribeirão Preto, USP, Ribeirão Preto, SP, Brazil Introduction: Metalloproteases are proteolytic enzymes present in several snake venoms. These proteins have important roles in the typical physiopathology of Bothrops envenomations. It is known that inhibitory compounds are present in serum, plasma and/or muscles of venomous snakes, and they are involved in a protective mechanism avoiding possible damages caused to the snake by its own venom. The aim of this work was the isolation and biochemical characterization of a novel metalloprotease inhibitor from Bothrops alternatus snake serum. Methods: Lyophilized B. alternatus snake serum was fractionated by four consecutive chromatographic steps: a DEAE-Sepharose, a size exclusion chromatography on Superdex200, an ion exchange chromatography on MonoQ and a reverse phase chromatography. The selection of the fraction of interest was made by the hemorrhagic activity, using a metalloprotease from B. atrox snake venom (Batroxase) as a positive control. The inhibitory potential was observed by the absence of formation of hemorrhagic halos in mice in comparison to the positive control. Results and conclusion: The obtained protein showed a single band with molecular mass around 66 kDa by SDS-PAGE, and 68.4 kDa by MALDI-TOF-MS and PI 4.0. The protein was named BaltMPI, and presented homology with a metalloprotease inhibitor from B. jararaca serum. The protein presented a inhibitory dose of 5.0 mg against Batroxase’s minimal hemorrhage dose (10 mg), with effective dose (ED50 ) of 0.982 mg. The characterization of inhibitory proteins as BaltMPI is very important as it could enable their use as antagonists of the damaging effects of snake venom metalloproteases. Financial support: CNPq, CAPES; FAPESP; NAP-TOXAN-USP. http://dx.doi.org/10.1016/j.toxlet.2014.06.309 P-1.130 The role of global histone modifications in Fumonisin B1-induced toxicity Duygu Sancak ∗ , Sibel Ozden Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Istanbul University, Istanbul, Turkey Fumonisin B1 (FB1) is the most prevalent member of a family of toxins produced by several species of Fusarium moulds which occur mainly in maize. Due to structural similarities with sphingolipid, FB1 exerts its toxic effects by disruption of sphingolipid biosynthesis, accumulation of sphinganine which can be highly cytotoxic and affects a wide variety of cellular system. FB1 was related to esophageal cancer in humans and was reported to be carcinogenic in rats. The mechanisms of underlying FB1 carcinogenicity are still unclear. Therefore, non-genotoxic (epigenetic) may play an important role in the mechanism of FB1 carcinogenicity. The aim of this study was to investigate dose and time dependent effects of FB1 on global histone modifications such as H3K9 methylation and H3K9 acetylation and the enzymes involved this mechanisms in rat kidney epithelial cells (NRK-52E). The increased level of global histone H3K9 di- and tri-methylation were observed in FB1-treated cells while level of global histone H3K9 acetylation was decreased. FB1 caused some changes on the activities of histone methyltransferase and histone acetyltransferase at high concentrations in NRK-52E cells. Further, the effects of trichostatin A, a histone deacetylase inhibitor, was investigated in NRK-52E cells and caused increase on