- Email: [email protected]
163. Antioxidant Effect of Camellia Sinensis (Green Tea) Extract Attenuate Acrylamide Induced Testicular Damage in Albino Rats
178
Abstracts Toxins 2012 / Toxicon 60 (2012) 95–248
162. Membrane-disturbing Properties of Urease and Derived Recombinant Peptides Anne H.S. Martinelli 1, Angela Piovesan 1, Karine Kappaun 1, Cristian Follmer 2, Jean-Louis Schwartz 3, Celia R. Carlini 1, 4 1
Graduate Program in Cellular and Molecular Biology – Centre of Biotechnology, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil 2 Dept. Physico-Chemistry, Inst. Chemistry, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil 3 Groupe d'étude des protéines membranaires, Department of Physiology, Université de Montreal, Montreal, Canada 4 Dept. Biophysics & Center of Biotechnology, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil E-mail address: [email protected] (C.R. Carlini).
Background: Ureases have long been known for their ureolytic activity, dependent on a Ni metallocenter active site. More recently novel toxic properties of ureases were discovered properties that are independent of the enzyme activity. While ureases of plant, fungi, and bacteria align with greater than 50% amino acid identity, toxicity may be due to more divergent domains. Plant ureases have potent toxicity against insects that are not affected by Bt toxins. In the case of jackbean (Canavalia ensiformis) urease (JBU), insecticidal activity relies mostly on an internal peptide (jaburetox-2) released upon ingestion by the insect's digestive enzymes. Modeling of Jaburetox-2 revealed a prominent b-hairpin motif consistent with an insecticidal activity based on either neurotoxicity or cell permeation. However, whole ureases display some entomotoxic properties not shared with its peptide. Here we describe membrane-disturbing properties of urease and of its insecticidal peptide and applied site-directed mutagenesis aiming to establish structure X activity relationships. Methods: the Planar Lipid Bilayer (PLB) and the carboxyfluorescein release assays were used to evaluate the membrane-disturbing properties of JBU and its derived peptides. Jaburetox-2 mutants were obtained by sitedirected mutagenesis (Stratagene): deletion of aminoacids 61-75 (Db-hairpin); deletion of N-terminal half (D1-44); deletion of the C-terminal half (D45-92). Results: JBU and Jaburetox-2 (5-10 nM) are able to insert into the PLB forming ionic channels. JBU's channels display four major conductance levels: 1730, 833, 625 and 352 pS and apparently have anion selectivity. Jaburetox-2 also forms channels with different conductance and kinetics. Jaburetox-2 (72 nM) induced leakage of carboxyfluorescein from large unilamellar vesicles composed of acidic lipids. The Db-hairpin mutant showed all the properties of the wild jaburetox-2 peptide, indicating that the b-hairpin motif is not relevant for the peptide's membrane-disturbing ability. On the other hand mutants corresponding to halves of the jaburetox-2 molecule (D1-44 and D45-92), although still active on the PLB and carboxyfluorescein leakage assays, showed much decreased activity, suggesting the presence of an active domain that was “split” between the two mutants. Conclusions: Membrane-disturbing and ion channel forming activities of urease and derived peptides may contribute to their diverse biological activities. These
domains are potential targets for manipulation to improve plant defense against herbivores and pathogens. Financial support by the Brazilian agencies CAPES, CNPq, FAPERGS and by the SEVE Centre, Quebec, Canada. Keywords: urease, jaburetox, membrane-disturbing 10.1016/j.toxicon.2012.04.163
163. Antioxidant Effect of Camellia Sinensis (Green Tea) Extract Attenuate Acrylamide Induced Testicular Damage in Albino Rats Yassa A. Heba 1, George M. Safaa 1, El Refaiy E. Abeer 2, Abd El Moneim M. Effat 3 1 Assiut University, Faculty of Medicine, Forensic and Clinical Toxicology Department, Egypt 2 Assiut University, Faculty of Medicine, Pathology Department, Egypt 3 Assiut University, Faculty of Medicine, Physiology Department, Egypt E-mail address: [email protected] (Y.A. Heba).
Fig. 1. Testosterone hormone level in different groups of animals.
Fig. 2. Effect of acrylamide and protective role of green tea on animal weight.
Abstracts Toxins 2012 / Toxicon 60 (2012) 95–248
179
Table (2): Effect of acrylamide and protective role of green tea on animal weight in mg: Animal At After 3 groups beginning weeks of the dose
After 6 weeks
After 9 weeks
After 12 weeks
G G G G G G
201.51.9 1791.4* 177.21.8** 196.81.8** 198.11.9** 199.82.0
212.82.2 180.31.5** 178.61.7** 106.71.9** 207.82.0** 109.72.1
223.62.1 1851.7** 183.21.4** 222.51.5** 224.21.9** 219.91.9
I II III IV V VI
1822.5 1802 1792.3 1812.7 1801.9 1771.8
1911.9 178.21.8* 176.51.8** 188.21.7** 189.92.1** 1902.1
Data are represented as meanS.D. of weight in mg (n ¼ 10). Asterisk indicates significant difference between groups and control, *p < 0.05; **p < 0.001.
Fig. 3. a) Testicles of control group (Group I). b) Group II showed minimal effects on testicular membrane when compared with the control group. c) Group III showed thickening of the tubular endothelium, degeneration of germ cells and formation of multinucleated giant cells. d) and e) Group IV and Group V showed no changes due to protective effects of green tea.
Background: Acrylamide is a proved toxin for testicular function, found in food when heated for long period of time. Green tea (Camellia sinensis) is a potent antioxidant; the aim of this study was to investigate the protective effect of green tea extract against the toxic effects of acrylamide in rat testes. Methods: acrylamide was administered orally to rats in different doses and also the extract of green tea was administered orally to different groups of animals in combination with the acrylamide. The weight of animals, testosterone hormone level and histopathological effect upon testicles were evaluated. Results: Testosterone hormone level in serum, and histopathological findings were significantly improved with the co administration of green tea extract with the acrylamide. Green tea extract reversed all the toxic effects of acrylamide even in high dose for long period (90 days). Conclusion: green tea extract is a potent antioxidant antidote for the acrylamide toxic effects upon testicular function.
Keywords: Camellia sinensis, testosterone, acrylamide 10.1016/j.toxicon.2012.04.164
164. Evaluation of Anticancer Activity Promoted by Molecules Contained in the Extracts of Thevetia peruviana Tamiris Caroline Barbon 1, Cássio Prinholato da Silva 2, Suely Vilela Sampaio 2, Mateus Amaral Baldo 1
Fig. 4. High power view of testis of Group III showing degeneration of germ cells and the formation of many multinucleated giant cells in atrophied seminiferous tubules (H&E X 400).
1 Laboratório de Produtos Naturais, Universidade Paulista, São José do Rio Pardo, SP Brazil 2 Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil E-mail address: [email protected] (M.A. Baldo).
Introduction: Thevetia peruviana is an evergreen shrub or a small plant in the Apocynaceae family, known for Table (1): Testosterone level in different studied groups. Groups
Serum testosterone level in ng/ml
No. of animals
No. of dead animals
Group Group Group Group Group Group
3.95 0 .87 2.510.65* 0.790.32** 3.920.33** 3.900.75** 3.940.88
10 10 10 10 10 10
0 0 1 0 0 0
I (control group) II (acrylamide 1/10 LD50)¼ 15mg/kg III (Acrylamide 1/5 LD50)¼ 30mg/kg IV (acrylamide 15 mg/kg þ 70 mg/kg green tea) V (acrylamide 30 mg/kg þ 70 mg/kg green tea) VI (green tea alone 70 mg/kg)
Data are represented as meanS.E. of testosterone hormone level (n ¼ 10). Asterisk indicates significant difference between groups, *p < 0.05; **p < 0.001.
Abstracts Toxins 2012 / Toxicon 60 (2012) 95–248
162. Membrane-disturbing Properties of Urease and Derived Recombinant Peptides Anne H.S. Martinelli 1, Angela Piovesan 1, Karine Kappaun 1, Cristian Follmer 2, Jean-Louis Schwartz 3, Celia R. Carlini 1, 4 1
Graduate Program in Cellular and Molecular Biology – Centre of Biotechnology, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil 2 Dept. Physico-Chemistry, Inst. Chemistry, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil 3 Groupe d'étude des protéines membranaires, Department of Physiology, Université de Montreal, Montreal, Canada 4 Dept. Biophysics & Center of Biotechnology, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil E-mail address: [email protected] (C.R. Carlini).
Background: Ureases have long been known for their ureolytic activity, dependent on a Ni metallocenter active site. More recently novel toxic properties of ureases were discovered properties that are independent of the enzyme activity. While ureases of plant, fungi, and bacteria align with greater than 50% amino acid identity, toxicity may be due to more divergent domains. Plant ureases have potent toxicity against insects that are not affected by Bt toxins. In the case of jackbean (Canavalia ensiformis) urease (JBU), insecticidal activity relies mostly on an internal peptide (jaburetox-2) released upon ingestion by the insect's digestive enzymes. Modeling of Jaburetox-2 revealed a prominent b-hairpin motif consistent with an insecticidal activity based on either neurotoxicity or cell permeation. However, whole ureases display some entomotoxic properties not shared with its peptide. Here we describe membrane-disturbing properties of urease and of its insecticidal peptide and applied site-directed mutagenesis aiming to establish structure X activity relationships. Methods: the Planar Lipid Bilayer (PLB) and the carboxyfluorescein release assays were used to evaluate the membrane-disturbing properties of JBU and its derived peptides. Jaburetox-2 mutants were obtained by sitedirected mutagenesis (Stratagene): deletion of aminoacids 61-75 (Db-hairpin); deletion of N-terminal half (D1-44); deletion of the C-terminal half (D45-92). Results: JBU and Jaburetox-2 (5-10 nM) are able to insert into the PLB forming ionic channels. JBU's channels display four major conductance levels: 1730, 833, 625 and 352 pS and apparently have anion selectivity. Jaburetox-2 also forms channels with different conductance and kinetics. Jaburetox-2 (72 nM) induced leakage of carboxyfluorescein from large unilamellar vesicles composed of acidic lipids. The Db-hairpin mutant showed all the properties of the wild jaburetox-2 peptide, indicating that the b-hairpin motif is not relevant for the peptide's membrane-disturbing ability. On the other hand mutants corresponding to halves of the jaburetox-2 molecule (D1-44 and D45-92), although still active on the PLB and carboxyfluorescein leakage assays, showed much decreased activity, suggesting the presence of an active domain that was “split” between the two mutants. Conclusions: Membrane-disturbing and ion channel forming activities of urease and derived peptides may contribute to their diverse biological activities. These
domains are potential targets for manipulation to improve plant defense against herbivores and pathogens. Financial support by the Brazilian agencies CAPES, CNPq, FAPERGS and by the SEVE Centre, Quebec, Canada. Keywords: urease, jaburetox, membrane-disturbing 10.1016/j.toxicon.2012.04.163
163. Antioxidant Effect of Camellia Sinensis (Green Tea) Extract Attenuate Acrylamide Induced Testicular Damage in Albino Rats Yassa A. Heba 1, George M. Safaa 1, El Refaiy E. Abeer 2, Abd El Moneim M. Effat 3 1 Assiut University, Faculty of Medicine, Forensic and Clinical Toxicology Department, Egypt 2 Assiut University, Faculty of Medicine, Pathology Department, Egypt 3 Assiut University, Faculty of Medicine, Physiology Department, Egypt E-mail address: [email protected] (Y.A. Heba).
Fig. 1. Testosterone hormone level in different groups of animals.
Fig. 2. Effect of acrylamide and protective role of green tea on animal weight.
Abstracts Toxins 2012 / Toxicon 60 (2012) 95–248
179
Table (2): Effect of acrylamide and protective role of green tea on animal weight in mg: Animal At After 3 groups beginning weeks of the dose
After 6 weeks
After 9 weeks
After 12 weeks
G G G G G G
201.51.9 1791.4* 177.21.8** 196.81.8** 198.11.9** 199.82.0
212.82.2 180.31.5** 178.61.7** 106.71.9** 207.82.0** 109.72.1
223.62.1 1851.7** 183.21.4** 222.51.5** 224.21.9** 219.91.9
I II III IV V VI
1822.5 1802 1792.3 1812.7 1801.9 1771.8
1911.9 178.21.8* 176.51.8** 188.21.7** 189.92.1** 1902.1
Data are represented as meanS.D. of weight in mg (n ¼ 10). Asterisk indicates significant difference between groups and control, *p < 0.05; **p < 0.001.
Fig. 3. a) Testicles of control group (Group I). b) Group II showed minimal effects on testicular membrane when compared with the control group. c) Group III showed thickening of the tubular endothelium, degeneration of germ cells and formation of multinucleated giant cells. d) and e) Group IV and Group V showed no changes due to protective effects of green tea.
Background: Acrylamide is a proved toxin for testicular function, found in food when heated for long period of time. Green tea (Camellia sinensis) is a potent antioxidant; the aim of this study was to investigate the protective effect of green tea extract against the toxic effects of acrylamide in rat testes. Methods: acrylamide was administered orally to rats in different doses and also the extract of green tea was administered orally to different groups of animals in combination with the acrylamide. The weight of animals, testosterone hormone level and histopathological effect upon testicles were evaluated. Results: Testosterone hormone level in serum, and histopathological findings were significantly improved with the co administration of green tea extract with the acrylamide. Green tea extract reversed all the toxic effects of acrylamide even in high dose for long period (90 days). Conclusion: green tea extract is a potent antioxidant antidote for the acrylamide toxic effects upon testicular function.
Keywords: Camellia sinensis, testosterone, acrylamide 10.1016/j.toxicon.2012.04.164
164. Evaluation of Anticancer Activity Promoted by Molecules Contained in the Extracts of Thevetia peruviana Tamiris Caroline Barbon 1, Cássio Prinholato da Silva 2, Suely Vilela Sampaio 2, Mateus Amaral Baldo 1
Fig. 4. High power view of testis of Group III showing degeneration of germ cells and the formation of many multinucleated giant cells in atrophied seminiferous tubules (H&E X 400).
1 Laboratório de Produtos Naturais, Universidade Paulista, São José do Rio Pardo, SP Brazil 2 Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil E-mail address: [email protected] (M.A. Baldo).
Introduction: Thevetia peruviana is an evergreen shrub or a small plant in the Apocynaceae family, known for Table (1): Testosterone level in different studied groups. Groups
Serum testosterone level in ng/ml
No. of animals
No. of dead animals
Group Group Group Group Group Group
3.95 0 .87 2.510.65* 0.790.32** 3.920.33** 3.900.75** 3.940.88
10 10 10 10 10 10
0 0 1 0 0 0
I (control group) II (acrylamide 1/10 LD50)¼ 15mg/kg III (Acrylamide 1/5 LD50)¼ 30mg/kg IV (acrylamide 15 mg/kg þ 70 mg/kg green tea) V (acrylamide 30 mg/kg þ 70 mg/kg green tea) VI (green tea alone 70 mg/kg)
Data are represented as meanS.E. of testosterone hormone level (n ¼ 10). Asterisk indicates significant difference between groups, *p < 0.05; **p < 0.001.