- Email: [email protected]
166. Toxins of Tityus Serrulatus Scorpion Venom Induce Inflammatory Mediators in vitro
180
Abstracts Toxins 2012 / Toxicon 60 (2012) 95–248
highly toxic properties, performed by molecules of cardenolides class. Besides toxicity, these molecules have shown a large potential therapeutic, such as antiparasitic, antimicrobial and also a significant anticancer activity. Cancer disease affect thousands of people and drug therapy is fundamental to increase survival or total cure of the disease. The aim of this study was to analyze the activity of extracts obtained from Thevetia peruviana in inhibition capacity of cell replication, important method in the therapy against the cancer. Method: The extract of Thevetia peruviana was obtained by cold maceration using methanol and subjected to reactions of Lieberman-Bouchard and Keddi. Those reactions were performed in the sample for identification of cardenolides. The cytotoxicity assays were performed using tumor cells line HL-60 (CCL-240, Acute Promyelocytic Leukemia Cells), HepG2 (HB-8065, Hepatocellular Carcinoma Human Cells), PC-12 (CRL-1721, Murine Pheochromocytoma Cells) obtained of ATCC (American Collection of Cell Culture). The cells were added in a 96-well plate and treated with different concentrations of extract (DCE) (5, 10, 25, 50, 100 and 200 mg/ mL) and incubated for 24 h. The positive control (PC) was done with cisplatin (1mg/mL). After the period, 10 mL of MTT were added to identify the viable cells and again subject in incubation for 3 h. at 37C , in 5% of CO2. After that, 100 mL of DMSO were added for solubilization of formazan crystals and the absorbance was measured. Results: The Lieberman-Bouchard and Keddi reactions showed positive results, confirming the presence of cardenolides in the extracts, and the different concentrations of extract inhibited the different tumor cells in the respective sequences : HEPG2 (PC: 18.1%) (DCE: 62.5%, 56.1%, 52.6%, 49.0%, 57.9%, 56.7%); HL60 (PC: 11.6%) (DCE: 90.9%, 61.5%, 55,5%, 52.6%, 38.7%, 26.3%); PC12 (PC: 20.7%) (DCE: 63.8%, 73.6%, 52.0%, 46.2%, 96.4%). Discussion: The test of cytotoxicity showed inhibition of cell replication in the three tumor cells, more effectively in the type HL-60, showing a dose-dependent correlation with major action in the concentration of 200 mg/mL. In HEP-G2 and in PC-12 the dose-dependence correlation was not observed but obtained significant inhibitions. Conclusions: A large variety of molecules presents in plants brings a huge arsenal of option to development of research in the many areas looking for therapeutics potential against diseases. Thevetia peruviana presents molecules that may be used to combat cancer. Keywords: Thevetia peruviana, anticancer activity, cardenolides 10.1016/j.toxicon.2012.04.165
M. Scorpions 165. Molecular Cloning, Expression and StructureFunction Analysis of Neopladine-2, an Antineoplastic Peptide from Tityus discrepans Scorpion Venom F. Olvera 1, G. D'Suze 2, A. Olvera 1, P. Diaz 2, A. Rosales 2, C. Sevcik 2, A. Alagón 1 1 Department of Molecular Medicine and Bioprocesses, Biotechnology Institute, National University of Mexico (UNAM), Mexico
2 Laboratory of Cellular Neuropharmacology, Biophysics and Biochemistry Center, Instituto Venezolano de Investigaciones Científicas (IVIC), Caracas, Venezuela E-mail address: [email protected] (A. Olvera).
Introduction: Tityus discrepans venom gland cDNA may have applications in treating human cancers. Methods: Total RNA was extracted from the venom glands of Tityus discrepans and cDNA was obtained by RT-PCR using primers complementary to the nucleotide sequences coding the first six amino acids of the mature protein. Results: The PCR products obtained were cloned and sequenced, establishing the complete bona fide sequence of the Neopladine 2. The deduced amino acid sequence comprises 245 residues. The protein was expressed in E. coli (XL1 Blue), as a fusion protein for subsequent H6 purification. The protein was obtained as inclusion bodies and folded in vitro to obtain a soluble protein. The folding yield was 80% obtaining about 9.6 mg/L of culture of soluble protein. Its primary structure shows moderate homology with ADAMTS Ca2þ-methalloproteinases and it was used to predict the tertiary structure by homology molecular simulation. The SWISS MODEL modeled protein was optimized with the molecular modeling program YASARA, with the YAMBER3 force field. Energy was minimized and binding energies were calculated. Future work will test the anti-neoplastic effect of recombinant and native N2 (1 mg/mL or w33 mM) on human breast carcinoma cell line SKBR3 (ATCC# Number: HTB-30#) and normal monkey kidney cell line MA104 (ATCC Number: CRL-2378.1). Financial support: Partially supported by FONACyT (Venezuela) and FONDEN grant to GDS. Supported in part by a grant provided by DGAPA/PAPIIT, IN-214211, UNAM. Keywords: recombinant, neopladine 2, Tityus discrepans 10.1016/j.toxicon.2012.04.166
166. Toxins of Tityus Serrulatus Scorpion Venom Induce Inflammatory Mediators in vitro Karina F. Zoccal 1, Claudia da S. Bitencourt 1, Carlos A. Sorgi 1, Karla de C.F. Bordon 2, Suely V. Sampaio 1, Eliane C. Arantes 2, Lúcia H. Faccioli 1 1 Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil 2 Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil E-mail address: [email protected] (K.F. Zoccal).
Background and Objectives: Tityus serrulatus (Ts) is responsible for the majority of cases of human poisoning by scorpions in Brazil. The specific signs of scorpion envenomation are directly related to the venom components. There are studies regarding venom actions, however little is known about the interactions of its toxins with immune cells. This study was designed to evaluate the ability of Ts1, Ts2 and Ts6, in combination or not with lipopolysaccharide (LPS), to induce production of cytokines, nitric oxide (NO) by immortalized alveolar macrophages (MH-S). Lipid bodies (LBs) formation was also investigated. LBs are lipid rich organelles distributed in the cytoplasm of most
Abstracts Toxins 2012 / Toxicon 60 (2012) 95–248
eukaryotic cells and are involved in a variety of functions such as lipid metabolism, cell signaling and inflammation. However, nothing is known about the formation and function of LBs in alveolar macrophages stimulated with Ts1, Ts2 and Ts6 from the venom of T. serrulatus escorpion. Methodology and Results: Ts1, Ts2 and Ts6 were not cytotoxic in all concentrations used in MH-S cells. NO concentration was measured by Greiss method and interleukin (IL)-6, IL-10 and tumor necrosis factor (TNF)-a by ELISA. NO, IL-6 and TNF-a production by MH-S cells, stimulated with Ts1 or Ts6, were enhanced under LPS prestimulation. On the other hand, Ts2 inhibited the release of these inflammatory mediators and increased IL-10 production. LBs formation increased after toxin stimulation compared to non-stimulated cells. Discussion and Conclusion: Our results demonstrated that, in vitro, individual scorpion toxins possess different properties. Ts1 and Ts6 presented similar effects that were opposite to Ts2 regarding to NO, TNF-a, IL-6 and IL-10. Ts1, Ts2 and Ts6 induced the formation of LBs, which could be related with eicosanoids production. We might suggest that production of cytokine and lipid mediators by toxinstimulated macrophages is independent of toxin ion channel interactions, since that Ts1 and Ts2 act on Naþ ion channels, and Ts6 act on Kþ ion channels.
181
tested in electrophysiological assays on a panel of six Kþ channels (Kv1.1-1.6) using the two-electrode voltage clamp technique. A cDNA library from the telson was constructed and specific screening of genes was conducted. Different algorithms and bioinformatics tools were used for the sequences analysis. Results: In the present study, we report for the first time, the molecular, biochemical and electrophysiological characterization of the components present in the soluble venom from M. gibbosus. Three new alpha-KTx peptides were found and called MegKTx1, MegKTx2 and MegKTx3 (Mesobuthus gibbosus, Kþ channel toxin number 1 to 3). Biochemical and molecular characterization of MegKTx peptides and genes shows a relation with toxins of three different alpha-KTx subfamilies. Conclusions: The exploration of the components present in the venom from M. gibbosus and the identification of gene sequences are important to understand the biological role of venom components that interact with Kþ channels. Consequently, this information may help in the dissection and knowledge of the noxious effects produced by this scorpion sting. References
167. Novel Potassium Channel Blocker Venom Peptides from Mesobuthus gibbosus (Scorpiones: Buthidae)
Tytgat et al., 1999 Trends Pharmacol Sci. 11:444-7. Srinivasan et al., 2002 J. Biol. Chem. 277:30040-47. Rodríguez de la Vega and Possani, 2004 Toxicon 43:865-75. Ceraretli and Ozkan 2010 Rev. Inst. Med. Trop. Sao Paulo 52(4):215-20. Adiguzel, 2010 J. Venom Anim. Toxins Incl. Trop. Dis 16:198-211.
Elia Diego-García 1, Steve Peigneur 1, Sarah Debaveye 1, Eveline Gheldof 1, Jan Tytgat 1, Figen Caliskan 2
Keywords: scorpion, alpha-KTx, toxin, gene 10.1016/j.toxicon.2012.04.168
Keywords: MH-S, Tityus serrulatus, inflammatory mediators 10.1016/j.toxicon.2012.04.167
1
Laboratory of Toxicology, University of Leuven (KUL), Leuven, Belgium Department of Biology, Faculty of Science and Art, Eskisehir Osmangazi University, Campus Meselik, Eskisehir, Turkey E-mail address: [email protected] (E. Diego-García). 2
Background: Scorpion toxins specific for potassium channels (KTx) have been classified on the basis of the alignment of Cys and other conserved residues into four families, known as alpha-, beta-, gamma-KTx [1] and kappaKTx [2]. The alpha-KTx family is considered the largest [3]. Until now, twenty-two subfamilies have been reported and several new peptides are continuously being discovered. Mesobuthus gibbosus (Brullé, 1832) belongs to the Buthidae family. This species is widely distributed in the Eastern Mediterranean region; the geographical range includes the Balkan Peninsula (Albania, Montenegro, Macedonia and Greece) and Anatolia (Turkey, except for the coast of the Black Sea). According to the epidemiological and clinical situation of scorpion envenomations in Turkey, M. gibbosus is one of the most important health-threatening scorpion species [4]. Despite the medical importance reported for M. gibbosus [5], there is no additional information of toxin and venom components to clarify the toxic effect of a M. gibbosus sting. Methods: Biochemical characterization was performed using different protocols and techniques following a bioassay-guided strategy (HPLC, mass spectrometry and EDMAN degradation sequencing). Venom fractions were
168. Tityus serrulatus Venom Induces a Higher Lung Inflammation in Mice Selected for Maximal Inflammatory Response Priscila G. Lara 1, Thaís R. Narcizo 1, Fernanda C.V. Portaro 2, Nancy Starobinas 1, Vera Aiello V 3, Luiz A. Benvenuti 3, Osvaldo A. Sant'Anna 2, Orlando G. Ribeiro 1, Mônica Spadafora-Ferreira 1 1
Laboratório de Imunogenética, Instituto Butantan, São Paulo, Brazil Laboratório de Imunoquímica, Instituto Butantan, São Paulo, Brazil 3 Laboratório de Patologia, Instituto do Coração (InCor), São Paulo, Brazil E-mail address: [email protected] (M. Spadafora-Ferreira). 2
Background: Tityus serrulatus is the main cause of scorpion envenomations in Brazil. Cardiovascular failure complicated by pulmonary edema is the main cause of death after severe envenomation. T. serrulatus venom (TsV) induces a systemic inflammatory response with the release of inflammatory mediators and cytokines both in patients and animal models. Lung alterations have been reported with the presence of inflammatory infiltrating cells and edema. The amount of venom inoculated, age, physical condition and genetic factors of the victims directly influence the severity of symptoms reported by patients. This study aimed to evaluate the action of TsV in the lung inflammation in strains of mice
Abstracts Toxins 2012 / Toxicon 60 (2012) 95–248
highly toxic properties, performed by molecules of cardenolides class. Besides toxicity, these molecules have shown a large potential therapeutic, such as antiparasitic, antimicrobial and also a significant anticancer activity. Cancer disease affect thousands of people and drug therapy is fundamental to increase survival or total cure of the disease. The aim of this study was to analyze the activity of extracts obtained from Thevetia peruviana in inhibition capacity of cell replication, important method in the therapy against the cancer. Method: The extract of Thevetia peruviana was obtained by cold maceration using methanol and subjected to reactions of Lieberman-Bouchard and Keddi. Those reactions were performed in the sample for identification of cardenolides. The cytotoxicity assays were performed using tumor cells line HL-60 (CCL-240, Acute Promyelocytic Leukemia Cells), HepG2 (HB-8065, Hepatocellular Carcinoma Human Cells), PC-12 (CRL-1721, Murine Pheochromocytoma Cells) obtained of ATCC (American Collection of Cell Culture). The cells were added in a 96-well plate and treated with different concentrations of extract (DCE) (5, 10, 25, 50, 100 and 200 mg/ mL) and incubated for 24 h. The positive control (PC) was done with cisplatin (1mg/mL). After the period, 10 mL of MTT were added to identify the viable cells and again subject in incubation for 3 h. at 37C , in 5% of CO2. After that, 100 mL of DMSO were added for solubilization of formazan crystals and the absorbance was measured. Results: The Lieberman-Bouchard and Keddi reactions showed positive results, confirming the presence of cardenolides in the extracts, and the different concentrations of extract inhibited the different tumor cells in the respective sequences : HEPG2 (PC: 18.1%) (DCE: 62.5%, 56.1%, 52.6%, 49.0%, 57.9%, 56.7%); HL60 (PC: 11.6%) (DCE: 90.9%, 61.5%, 55,5%, 52.6%, 38.7%, 26.3%); PC12 (PC: 20.7%) (DCE: 63.8%, 73.6%, 52.0%, 46.2%, 96.4%). Discussion: The test of cytotoxicity showed inhibition of cell replication in the three tumor cells, more effectively in the type HL-60, showing a dose-dependent correlation with major action in the concentration of 200 mg/mL. In HEP-G2 and in PC-12 the dose-dependence correlation was not observed but obtained significant inhibitions. Conclusions: A large variety of molecules presents in plants brings a huge arsenal of option to development of research in the many areas looking for therapeutics potential against diseases. Thevetia peruviana presents molecules that may be used to combat cancer. Keywords: Thevetia peruviana, anticancer activity, cardenolides 10.1016/j.toxicon.2012.04.165
M. Scorpions 165. Molecular Cloning, Expression and StructureFunction Analysis of Neopladine-2, an Antineoplastic Peptide from Tityus discrepans Scorpion Venom F. Olvera 1, G. D'Suze 2, A. Olvera 1, P. Diaz 2, A. Rosales 2, C. Sevcik 2, A. Alagón 1 1 Department of Molecular Medicine and Bioprocesses, Biotechnology Institute, National University of Mexico (UNAM), Mexico
2 Laboratory of Cellular Neuropharmacology, Biophysics and Biochemistry Center, Instituto Venezolano de Investigaciones Científicas (IVIC), Caracas, Venezuela E-mail address: [email protected] (A. Olvera).
Introduction: Tityus discrepans venom gland cDNA may have applications in treating human cancers. Methods: Total RNA was extracted from the venom glands of Tityus discrepans and cDNA was obtained by RT-PCR using primers complementary to the nucleotide sequences coding the first six amino acids of the mature protein. Results: The PCR products obtained were cloned and sequenced, establishing the complete bona fide sequence of the Neopladine 2. The deduced amino acid sequence comprises 245 residues. The protein was expressed in E. coli (XL1 Blue), as a fusion protein for subsequent H6 purification. The protein was obtained as inclusion bodies and folded in vitro to obtain a soluble protein. The folding yield was 80% obtaining about 9.6 mg/L of culture of soluble protein. Its primary structure shows moderate homology with ADAMTS Ca2þ-methalloproteinases and it was used to predict the tertiary structure by homology molecular simulation. The SWISS MODEL modeled protein was optimized with the molecular modeling program YASARA, with the YAMBER3 force field. Energy was minimized and binding energies were calculated. Future work will test the anti-neoplastic effect of recombinant and native N2 (1 mg/mL or w33 mM) on human breast carcinoma cell line SKBR3 (ATCC# Number: HTB-30#) and normal monkey kidney cell line MA104 (ATCC Number: CRL-2378.1). Financial support: Partially supported by FONACyT (Venezuela) and FONDEN grant to GDS. Supported in part by a grant provided by DGAPA/PAPIIT, IN-214211, UNAM. Keywords: recombinant, neopladine 2, Tityus discrepans 10.1016/j.toxicon.2012.04.166
166. Toxins of Tityus Serrulatus Scorpion Venom Induce Inflammatory Mediators in vitro Karina F. Zoccal 1, Claudia da S. Bitencourt 1, Carlos A. Sorgi 1, Karla de C.F. Bordon 2, Suely V. Sampaio 1, Eliane C. Arantes 2, Lúcia H. Faccioli 1 1 Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil 2 Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil E-mail address: [email protected] (K.F. Zoccal).
Background and Objectives: Tityus serrulatus (Ts) is responsible for the majority of cases of human poisoning by scorpions in Brazil. The specific signs of scorpion envenomation are directly related to the venom components. There are studies regarding venom actions, however little is known about the interactions of its toxins with immune cells. This study was designed to evaluate the ability of Ts1, Ts2 and Ts6, in combination or not with lipopolysaccharide (LPS), to induce production of cytokines, nitric oxide (NO) by immortalized alveolar macrophages (MH-S). Lipid bodies (LBs) formation was also investigated. LBs are lipid rich organelles distributed in the cytoplasm of most
Abstracts Toxins 2012 / Toxicon 60 (2012) 95–248
eukaryotic cells and are involved in a variety of functions such as lipid metabolism, cell signaling and inflammation. However, nothing is known about the formation and function of LBs in alveolar macrophages stimulated with Ts1, Ts2 and Ts6 from the venom of T. serrulatus escorpion. Methodology and Results: Ts1, Ts2 and Ts6 were not cytotoxic in all concentrations used in MH-S cells. NO concentration was measured by Greiss method and interleukin (IL)-6, IL-10 and tumor necrosis factor (TNF)-a by ELISA. NO, IL-6 and TNF-a production by MH-S cells, stimulated with Ts1 or Ts6, were enhanced under LPS prestimulation. On the other hand, Ts2 inhibited the release of these inflammatory mediators and increased IL-10 production. LBs formation increased after toxin stimulation compared to non-stimulated cells. Discussion and Conclusion: Our results demonstrated that, in vitro, individual scorpion toxins possess different properties. Ts1 and Ts6 presented similar effects that were opposite to Ts2 regarding to NO, TNF-a, IL-6 and IL-10. Ts1, Ts2 and Ts6 induced the formation of LBs, which could be related with eicosanoids production. We might suggest that production of cytokine and lipid mediators by toxinstimulated macrophages is independent of toxin ion channel interactions, since that Ts1 and Ts2 act on Naþ ion channels, and Ts6 act on Kþ ion channels.
181
tested in electrophysiological assays on a panel of six Kþ channels (Kv1.1-1.6) using the two-electrode voltage clamp technique. A cDNA library from the telson was constructed and specific screening of genes was conducted. Different algorithms and bioinformatics tools were used for the sequences analysis. Results: In the present study, we report for the first time, the molecular, biochemical and electrophysiological characterization of the components present in the soluble venom from M. gibbosus. Three new alpha-KTx peptides were found and called MegKTx1, MegKTx2 and MegKTx3 (Mesobuthus gibbosus, Kþ channel toxin number 1 to 3). Biochemical and molecular characterization of MegKTx peptides and genes shows a relation with toxins of three different alpha-KTx subfamilies. Conclusions: The exploration of the components present in the venom from M. gibbosus and the identification of gene sequences are important to understand the biological role of venom components that interact with Kþ channels. Consequently, this information may help in the dissection and knowledge of the noxious effects produced by this scorpion sting. References
167. Novel Potassium Channel Blocker Venom Peptides from Mesobuthus gibbosus (Scorpiones: Buthidae)
Tytgat et al., 1999 Trends Pharmacol Sci. 11:444-7. Srinivasan et al., 2002 J. Biol. Chem. 277:30040-47. Rodríguez de la Vega and Possani, 2004 Toxicon 43:865-75. Ceraretli and Ozkan 2010 Rev. Inst. Med. Trop. Sao Paulo 52(4):215-20. Adiguzel, 2010 J. Venom Anim. Toxins Incl. Trop. Dis 16:198-211.
Elia Diego-García 1, Steve Peigneur 1, Sarah Debaveye 1, Eveline Gheldof 1, Jan Tytgat 1, Figen Caliskan 2
Keywords: scorpion, alpha-KTx, toxin, gene 10.1016/j.toxicon.2012.04.168
Keywords: MH-S, Tityus serrulatus, inflammatory mediators 10.1016/j.toxicon.2012.04.167
1
Laboratory of Toxicology, University of Leuven (KUL), Leuven, Belgium Department of Biology, Faculty of Science and Art, Eskisehir Osmangazi University, Campus Meselik, Eskisehir, Turkey E-mail address: [email protected] (E. Diego-García). 2
Background: Scorpion toxins specific for potassium channels (KTx) have been classified on the basis of the alignment of Cys and other conserved residues into four families, known as alpha-, beta-, gamma-KTx [1] and kappaKTx [2]. The alpha-KTx family is considered the largest [3]. Until now, twenty-two subfamilies have been reported and several new peptides are continuously being discovered. Mesobuthus gibbosus (Brullé, 1832) belongs to the Buthidae family. This species is widely distributed in the Eastern Mediterranean region; the geographical range includes the Balkan Peninsula (Albania, Montenegro, Macedonia and Greece) and Anatolia (Turkey, except for the coast of the Black Sea). According to the epidemiological and clinical situation of scorpion envenomations in Turkey, M. gibbosus is one of the most important health-threatening scorpion species [4]. Despite the medical importance reported for M. gibbosus [5], there is no additional information of toxin and venom components to clarify the toxic effect of a M. gibbosus sting. Methods: Biochemical characterization was performed using different protocols and techniques following a bioassay-guided strategy (HPLC, mass spectrometry and EDMAN degradation sequencing). Venom fractions were
168. Tityus serrulatus Venom Induces a Higher Lung Inflammation in Mice Selected for Maximal Inflammatory Response Priscila G. Lara 1, Thaís R. Narcizo 1, Fernanda C.V. Portaro 2, Nancy Starobinas 1, Vera Aiello V 3, Luiz A. Benvenuti 3, Osvaldo A. Sant'Anna 2, Orlando G. Ribeiro 1, Mônica Spadafora-Ferreira 1 1
Laboratório de Imunogenética, Instituto Butantan, São Paulo, Brazil Laboratório de Imunoquímica, Instituto Butantan, São Paulo, Brazil 3 Laboratório de Patologia, Instituto do Coração (InCor), São Paulo, Brazil E-mail address: [email protected] (M. Spadafora-Ferreira). 2
Background: Tityus serrulatus is the main cause of scorpion envenomations in Brazil. Cardiovascular failure complicated by pulmonary edema is the main cause of death after severe envenomation. T. serrulatus venom (TsV) induces a systemic inflammatory response with the release of inflammatory mediators and cytokines both in patients and animal models. Lung alterations have been reported with the presence of inflammatory infiltrating cells and edema. The amount of venom inoculated, age, physical condition and genetic factors of the victims directly influence the severity of symptoms reported by patients. This study aimed to evaluate the action of TsV in the lung inflammation in strains of mice