- Email: [email protected]
243 Effect of crotapotin on the biological activity of D49 and K49 bothrops PLA2
s68
Poster Session P10. Natural toxins
sinergistic effect of these ether oils and derivative of fulerene (C60 (OOC-CHOH-CH2 -CH3 )n ). Laurel leaves and berries ether oils increased both the production of OH• radicals and intensity of lipid peroxidation, but this increase was lower as mass concentration of ether oils increased. Fulerene derivative used alone increased the production of OH• radicals and the intensity of lipid peroxidation (higher percentage than production of OH• radicals). Combination of laurel leaves and berries ether oil and fulerene derivative increased OH• production even more. Intensity of lipid peroxidation remained unchanged in combination of fulerene derivative and laurel ether oils. 241
STUDY EFFECT PEGANUM HARMALA PLANT ON SKELETAL SYSTEM
M.T. Goghataei, F. Kermanian, M. Mehdizadeh. Anatomy Department, Iran University, Tehran, Iran Peganum Harmala is popular plant in traditional medicine. It used for the treatment of asthma, parasitical and microbial infections, postpartum hemorrhage and etc. In order to study the side effects of PJHL in pregnant mice such as growth retardation and abortion, we injected P.H. intraperitoneally to the pregnant mice in three doses: 6, 15,30 mg/kg/day from the first up to the lo"h day of pregnancy, then we killed the mice in 15–19" pregnancy days. After Laparatomy we extract fetuses and measured their height and weight. Then the fetuses were stained with Alizarin Red and observed with stereomicroscope for skeletal anomalies. Conclusions showed that there were no skeletal anomalies. But in comparison with control group, absorption and decrease of height and weight was observed. Since the most absorption of the fetuses was in the last days of pregnancy (18–19th), we believe that P.H. causes abortion by means of affecting on growth and development offetus. 242
MICROCYSTIN-LR CAUSES REORGANIZATION OF ACTIN FILAMENTS AND MICROTUBULES IN RABBIT WHOLE EMBRYO CULTURES
M.C. Žužek 1 , M. Kosec 1 , J. Mrkun 1 , D. Šuput 3 , B. Sedmak 2 , R. Frangež 1 . 1 Veterinary faculty, Institute of physiology, pharmacology and toxicology, University of Ljubljana, Slovenia, 2 National Institute of Biology, University of Ljubljana, Slovenia, 3 Medical faculty, University of Ljubljana, Slovenia Microcystins are a group of cyclic heptapeptide hepatotoxins, produced by different cyanobacterial genera. Cyanobacteria are inhabitants of terrestrial, fresh and brackish water. Microcystin-LR (MC-LR), the most investigated microcystin, is produced by different Microcystis species. MC-LR is inhibitor of cellular protein phosphatases types 1 and 2A. The consequences of this effect are evident as alteration and redistribution of intermediate filaments, microtubules and microfilaments. The purpose of this study was to establish the in vitro effect of MC-LR on microtubules and actin filaments of whole embryo cell cultures. The embryos were harvested from super ovulated four months old female rabbits (New Zealand White) and cultivated in the presence of different final concentrations of microcystin (10 µM, 20 µM and 100 µM) for a period of 24 h. Whole embryo cell cultures were fixed, stained with fluorescent dye Rhodamine phaloidin for actin filaments and immunostained with anti-α-tubulin, mouse monoclonal antibodies and Alexa Fluor® fluorescent dye conjugate secondary antibodies for microtubules. Confocal laser microscopy was used for visualization of changes in organization of cytoskeleton. Low concentrations (10 µM and 20 µM) of MC-LR caused reorganization of microtubules and actin filaments in all whole embryo cell cultures without evident morphological changes. 100 µM MC-LR caused morphological changes which resulted in rounding of cells and loss of cell-cell adhesion, leading to detachment and dispersion of the cells. Our results confirmed that MC-LR affects actin and microtubule network distribution in whole embryo cell cultures in vitro.
243
EFFECT OF CROTAPOTIN ON THE BIOLOGICAL ACTIVITY OF D49 AND K49 BOTHROPS PLA2
E.C. Arantes 1 , A.L. Cecchini 1,2 , A.M. Soares 3 , R. Cecchini 4 , C.A. Vieira 2 , J.R. Giglio 2 . 1 Depto. Física e Química, FCFRP-USP; 2 Depto. Bioquímica e Imunologia, FMRP-USP; 3 Unidade de Biotecnologia, UNAERP; 4 Depto. Patologia Geral, Centro de Ciências Biológicas, UEL; 5 Depto. Química, FFCLRP, USP; Ribeirão Preto, SP, Brazil Myonecrosis is the most striking local effect caused by Bothrops snake venoms, while the ischaemia evoked by edema may aggravate the venom-induced lesion. These effects are partially due to phospholipase A2 toxins (PLA2 ). Some of them are catalytically active, (D49-PLA2 s), whereas others have little or no enzymatic activity due to a substitution of aspartic acid for lysine at position 49 (L49-PLA2 ), althoug they are very active as myonecrosis inducers. Crotoxin from Crotalus durissus terrificus venom is made up of two non-identical subunits: a basic PLA2 subunit and an acidic, non-toxic and non-enzymatic subunit, crotapotin, which prevents the PLA2 subunit from binding to non-specific sites, increasing its toxicity. In addition, crotapotin inhibits the edema induced by snake venoms or carrageenin, probably by interacting with PLA2 s secreted during the inflammatory process. We have investigated the effect of crotapotin on mouse paw edema and myonecrosis induced by isolated PLA2 s (D49 and K49) from three different species of Bothrops snake venom (BthTX-I and BthTX-II – B. jararacussu, PrTX-I and PrTX-III – B. pirajai and MjTX-II – B. moojeni). We also assayed the enzymatic activity of the PLA2 s in the presence and absence of crotapotin. The combination of crotapotin with PLA2 (2:1, w/w) prior to injection resulted in great reductions in edema (the thickness of the mouse paw was used as an index of edema) and myonecrosis. Crotapotin (100 µg) inhibited around 40–50% the myotoxicity caused by PLA2 s (50 µg), evidenced by decrease in creatine kinase activity (CK-UV kit from Sigma Chemical Co.). It inhibited significantly the edema induce by BthTX-I (23% inhibition), BthTX-II (27%), PrTX-I (25%), PrTX-III (35%) and MjTX-II (10%). Saline and crotapotin alone were used as controls. We could not detect any significant inhibition of D49-PLA2 phospholipase activity, indicating that the interaction with crotapotin does not change the catalytic site. Support: FAPESP, CNPq. 244
NORADRENERGIC AND NITRERGIC EFFECTS INDUCED BY Tityus serrulatus VENOM ON THE RAT ISOLATED RETRACTOR PENIS MUSCLE.
E.C. Arantes 1 , J.H.G.G. Bomfim 1 , M.A.F. de Godoy 2 , J.R. Giglio 3 , A.M. de Oliveira 1 . 1 Depto. Física e Química, FCFRP-USP, 2 Depto Farmacologia, FMRP-USP, 3 Depto. Bioquímica e Imunologia, FMRP-USP, Ribeirão Preto, SP, Brazil. The main toxins from Tityus serrulatus venom (TsV) affect voltagegated sodium channels inducing the release of neurotransmitters from sympathetic and parasympathetic nerve terminals. Some evidence regarding the participation of nitrergic response on the effects induced by TsV has been provided by functional studies on rabbit corpus cavernosum. Thus, the aim of the present study was to investigate the participation of noradrenergic and nitrergic components on the responses of rat retractor penis muscle (RPM) to TsV, fractions X, XI, XIIa, XIIb and TsTX-I. RPM was isolated and mounted under 0.6 g isotonic resting tension in 5 mL organ bath containing physiological salt solution. Contractions and relaxations were recorded as changes in the displacement (mm) from baseline and expressed as percentage of the maximum effect induced by KCl (90 mM) or of the maximum relaxant response, respectively. The relaxant responses were performed by contracting the muscle with bethanechol (100 µM), in the presence of prazosin (1 µM, 20 min) and guanetidine (30 µM, 10 min), associated or not with Nω -nitro-L-arginine methyl ester (L-NAME, 100 µM, 30 min) or tetrodotoxin (5 µM, 30 min). TsV and fractions (0.03 – 100 µg/mL) induced concentrationdependent contractile responses, while TsTX-I did not. Prazosin, guanetidine and tetrodotoxin completely abolished the contractile responses. TsV or fractions did not affect the cholinergic innervation. These results indicate that the contractile responses are due to the
Poster Session P10. Natural toxins
sinergistic effect of these ether oils and derivative of fulerene (C60 (OOC-CHOH-CH2 -CH3 )n ). Laurel leaves and berries ether oils increased both the production of OH• radicals and intensity of lipid peroxidation, but this increase was lower as mass concentration of ether oils increased. Fulerene derivative used alone increased the production of OH• radicals and the intensity of lipid peroxidation (higher percentage than production of OH• radicals). Combination of laurel leaves and berries ether oil and fulerene derivative increased OH• production even more. Intensity of lipid peroxidation remained unchanged in combination of fulerene derivative and laurel ether oils. 241
STUDY EFFECT PEGANUM HARMALA PLANT ON SKELETAL SYSTEM
M.T. Goghataei, F. Kermanian, M. Mehdizadeh. Anatomy Department, Iran University, Tehran, Iran Peganum Harmala is popular plant in traditional medicine. It used for the treatment of asthma, parasitical and microbial infections, postpartum hemorrhage and etc. In order to study the side effects of PJHL in pregnant mice such as growth retardation and abortion, we injected P.H. intraperitoneally to the pregnant mice in three doses: 6, 15,30 mg/kg/day from the first up to the lo"h day of pregnancy, then we killed the mice in 15–19" pregnancy days. After Laparatomy we extract fetuses and measured their height and weight. Then the fetuses were stained with Alizarin Red and observed with stereomicroscope for skeletal anomalies. Conclusions showed that there were no skeletal anomalies. But in comparison with control group, absorption and decrease of height and weight was observed. Since the most absorption of the fetuses was in the last days of pregnancy (18–19th), we believe that P.H. causes abortion by means of affecting on growth and development offetus. 242
MICROCYSTIN-LR CAUSES REORGANIZATION OF ACTIN FILAMENTS AND MICROTUBULES IN RABBIT WHOLE EMBRYO CULTURES
M.C. Žužek 1 , M. Kosec 1 , J. Mrkun 1 , D. Šuput 3 , B. Sedmak 2 , R. Frangež 1 . 1 Veterinary faculty, Institute of physiology, pharmacology and toxicology, University of Ljubljana, Slovenia, 2 National Institute of Biology, University of Ljubljana, Slovenia, 3 Medical faculty, University of Ljubljana, Slovenia Microcystins are a group of cyclic heptapeptide hepatotoxins, produced by different cyanobacterial genera. Cyanobacteria are inhabitants of terrestrial, fresh and brackish water. Microcystin-LR (MC-LR), the most investigated microcystin, is produced by different Microcystis species. MC-LR is inhibitor of cellular protein phosphatases types 1 and 2A. The consequences of this effect are evident as alteration and redistribution of intermediate filaments, microtubules and microfilaments. The purpose of this study was to establish the in vitro effect of MC-LR on microtubules and actin filaments of whole embryo cell cultures. The embryos were harvested from super ovulated four months old female rabbits (New Zealand White) and cultivated in the presence of different final concentrations of microcystin (10 µM, 20 µM and 100 µM) for a period of 24 h. Whole embryo cell cultures were fixed, stained with fluorescent dye Rhodamine phaloidin for actin filaments and immunostained with anti-α-tubulin, mouse monoclonal antibodies and Alexa Fluor® fluorescent dye conjugate secondary antibodies for microtubules. Confocal laser microscopy was used for visualization of changes in organization of cytoskeleton. Low concentrations (10 µM and 20 µM) of MC-LR caused reorganization of microtubules and actin filaments in all whole embryo cell cultures without evident morphological changes. 100 µM MC-LR caused morphological changes which resulted in rounding of cells and loss of cell-cell adhesion, leading to detachment and dispersion of the cells. Our results confirmed that MC-LR affects actin and microtubule network distribution in whole embryo cell cultures in vitro.
243
EFFECT OF CROTAPOTIN ON THE BIOLOGICAL ACTIVITY OF D49 AND K49 BOTHROPS PLA2
E.C. Arantes 1 , A.L. Cecchini 1,2 , A.M. Soares 3 , R. Cecchini 4 , C.A. Vieira 2 , J.R. Giglio 2 . 1 Depto. Física e Química, FCFRP-USP; 2 Depto. Bioquímica e Imunologia, FMRP-USP; 3 Unidade de Biotecnologia, UNAERP; 4 Depto. Patologia Geral, Centro de Ciências Biológicas, UEL; 5 Depto. Química, FFCLRP, USP; Ribeirão Preto, SP, Brazil Myonecrosis is the most striking local effect caused by Bothrops snake venoms, while the ischaemia evoked by edema may aggravate the venom-induced lesion. These effects are partially due to phospholipase A2 toxins (PLA2 ). Some of them are catalytically active, (D49-PLA2 s), whereas others have little or no enzymatic activity due to a substitution of aspartic acid for lysine at position 49 (L49-PLA2 ), althoug they are very active as myonecrosis inducers. Crotoxin from Crotalus durissus terrificus venom is made up of two non-identical subunits: a basic PLA2 subunit and an acidic, non-toxic and non-enzymatic subunit, crotapotin, which prevents the PLA2 subunit from binding to non-specific sites, increasing its toxicity. In addition, crotapotin inhibits the edema induced by snake venoms or carrageenin, probably by interacting with PLA2 s secreted during the inflammatory process. We have investigated the effect of crotapotin on mouse paw edema and myonecrosis induced by isolated PLA2 s (D49 and K49) from three different species of Bothrops snake venom (BthTX-I and BthTX-II – B. jararacussu, PrTX-I and PrTX-III – B. pirajai and MjTX-II – B. moojeni). We also assayed the enzymatic activity of the PLA2 s in the presence and absence of crotapotin. The combination of crotapotin with PLA2 (2:1, w/w) prior to injection resulted in great reductions in edema (the thickness of the mouse paw was used as an index of edema) and myonecrosis. Crotapotin (100 µg) inhibited around 40–50% the myotoxicity caused by PLA2 s (50 µg), evidenced by decrease in creatine kinase activity (CK-UV kit from Sigma Chemical Co.). It inhibited significantly the edema induce by BthTX-I (23% inhibition), BthTX-II (27%), PrTX-I (25%), PrTX-III (35%) and MjTX-II (10%). Saline and crotapotin alone were used as controls. We could not detect any significant inhibition of D49-PLA2 phospholipase activity, indicating that the interaction with crotapotin does not change the catalytic site. Support: FAPESP, CNPq. 244
NORADRENERGIC AND NITRERGIC EFFECTS INDUCED BY Tityus serrulatus VENOM ON THE RAT ISOLATED RETRACTOR PENIS MUSCLE.
E.C. Arantes 1 , J.H.G.G. Bomfim 1 , M.A.F. de Godoy 2 , J.R. Giglio 3 , A.M. de Oliveira 1 . 1 Depto. Física e Química, FCFRP-USP, 2 Depto Farmacologia, FMRP-USP, 3 Depto. Bioquímica e Imunologia, FMRP-USP, Ribeirão Preto, SP, Brazil. The main toxins from Tityus serrulatus venom (TsV) affect voltagegated sodium channels inducing the release of neurotransmitters from sympathetic and parasympathetic nerve terminals. Some evidence regarding the participation of nitrergic response on the effects induced by TsV has been provided by functional studies on rabbit corpus cavernosum. Thus, the aim of the present study was to investigate the participation of noradrenergic and nitrergic components on the responses of rat retractor penis muscle (RPM) to TsV, fractions X, XI, XIIa, XIIb and TsTX-I. RPM was isolated and mounted under 0.6 g isotonic resting tension in 5 mL organ bath containing physiological salt solution. Contractions and relaxations were recorded as changes in the displacement (mm) from baseline and expressed as percentage of the maximum effect induced by KCl (90 mM) or of the maximum relaxant response, respectively. The relaxant responses were performed by contracting the muscle with bethanechol (100 µM), in the presence of prazosin (1 µM, 20 min) and guanetidine (30 µM, 10 min), associated or not with Nω -nitro-L-arginine methyl ester (L-NAME, 100 µM, 30 min) or tetrodotoxin (5 µM, 30 min). TsV and fractions (0.03 – 100 µg/mL) induced concentrationdependent contractile responses, while TsTX-I did not. Prazosin, guanetidine and tetrodotoxin completely abolished the contractile responses. TsV or fractions did not affect the cholinergic innervation. These results indicate that the contractile responses are due to the