Toxicity intraperitoneal and intragastric route of Bacillus thuringiensis and Melia azedarach in mice

Toxicidade via intragástrica e intraperitoneal de Bacillus thuringiensis e Melia azedarach, em camundongos

Abstracts

The aim of this investigation was the assessment of toxicity of two new isolates of Bacillus thuringiensis, and the aqueous extract of Melia azedarach through in vivo assays in CF1 mice. Bt 1958-2, Bt 2014-2 and the BTh Thuricide 63 standard isolates were grown in liquid usual glicosed medium, and Cry proteins were purified by centrifugation on a sucrose gradient. The supernatant was autoclaved at 121º C, 15min. to maintain the exotoxins. Dehydrated leaves of M. azedarach were used to prepare a 10% aqueous extract. Mice were treated either orally or intraperitoneally with a whole bacterial suspension (1.10(10) UFC/mL), a culture supernatant or purified crystal protein (50 µg/mL), and with the plant extract (50 µg/mL). The stomachs of the mice were collected and observed in stereomicroscopy, and the stomach contents were analyzed in 10% SDS-PAGE. Results showed that none of the oral treatments were toxic to mice, but intraperitoneal bacterial suspensions were lethal to the animals 6 - 24 hours after injection. In conclusion, the Cry proteins of the new B. thuringiensis isolates must be evaluated for their use as tools in the biotechnology field, since they do not show toxicity against mammals, intragastrically or peritoneally, just like the M. azedarach aqueous extract (10%), with those being indicated for the biological control of pest insects.

Bacterium; mammalian; Meliaceae; toxicology

O objetivo deste estudo foi a avaliação da toxicidade de dois novos isolados de Bacillus thuringiensis e o extrato aquoso de Melia azedarach, através de ensaios in vivo em camundongos CF1. Os isolados Bt 1958-2, Bt 2014-2 e o isolado padrão BTH Thuricide 63 foram cultivados em meio usual glicosado, e as proteínas Cry foram purificadas por centrifugação em gradiente de sacarose. O sobrenadante foi tratado em autoclave a 121º C, 15 min para manter as exotoxinas. As folhas desidratadas de M. azedarach foram utilizadas para preparar um extrato aquoso a 10%. Camundongos foram tratados, via oral ou por via intraperitoneal, com a suspensão bacteriana (1.10(10) UFC/mL), o sobrenadante de cultura ou a proteína do cristal purificada (50 µg/mL), e com o extrato da planta (50 µg/mL). Os estômagos dos ratos foram coletados e observados em estereomicroscópio e os conteúdos estomacais foram analisados em SDS-PAGE a 10%. Os resultados mostraram que nenhum dos tratamentos orais foram tóxicos para os camundongos, mas, via intraperitoneal, as suspensões bacterianas foram letais para os animais entre 6 e 24 horas após a injeção. Em conclusão, as proteínas Cry dos novos isolados de B. thuringiensis devem ser avaliadas para sua utilização como ferramenta no campo da biotecnologia, uma vez que elas não mostram toxicidade contra mamíferos, intragástrica ou intraperitoneal, assim como o extrato aquoso (10%) de M. azedarach, podendo ser indicado para o controle biológico de insetos-praga.

Bactéria; mamíferos; Meliaceae; toxicologia

• AGAISSE, H.; GOMINET, M.; OKSTAD, O.A.; KOLSTO, A.; LERECLUS, D. PlcR is a pleiotropic regulator of extracellular virulence factor gene expression in Bacillus thuringiensis Molecular Microbiology, v.32, n.5, p.1043-1053, 1999.
• AZEVEDO, J.L.; ARAÚJO, W.L. Genetically modified crops: environmental and human health concerns. Mutation Research, v.544, p.223-233, 2003.
• BARJAC, H.; LECADET, M.M. Dosage biochimique d'exotoxine thermostable de Bacillus thuringiensis d'après i'nhibition d' arn-polymerases bacteriennes. Comptes Rendus de l'Academie des Sciences, v.282, p.2119-2122, 1976.
• BETZ, F.S.; HAMMOND, B.G.; FUCHS, R.L. Safety and advantages of Bacillus thuringiensis: protected plants to control insect pests. Regulatory Toxicology and Pharmacology, v.32, p.156-173, 2000.
• BISHOP, A.H.; JOHNSON, C.; PERANI, M. The safety of Bacillus thuringiensis to mammals investigated by oral and subcutaneous dosage. World Journal of Microbiology and Biotecnology, v.15, p.375-380, 1999.
• BRADFORD, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, v.72, p.248-254, 1976.
• BRUNHEROTTO, R.; VENDRAMIM, J.D. Bioatividade de extratos aquosos de Melia azedarach L. sobre o desenvolvimento de Tuta absoluta (Lepidoptera: Gelechiidae) em tomateiro. Neotropical Entomology, v.30, n.3, p.455-459, 2001.
• CAPALBO, D.M.F.; VILAS-BÔAS, G.T.; SUZUKI, M.T. Bacillus thuringiensis Biotecnologia Ciência & Desenvolvimento, v.34, p.78-85, 2005.
• CARPINELLA, M.C.; DEFAGO, M.T.; VALLADARES, G.; PALACIOS, S.M. Antifeedant and inseticide properties of a limnoid from Melia azedarach (Meliaceae) with potencial use for pest management. Journal of Agricultural and Food Chemistry, v.51, p.369-374, 2003.
• CORREA, R.F.T.; ARDISSON-ARAÚJO, D.M.P.; MONNERAT, R.G.; RIBEIRO, B.M. Cytotoxicity Analysis of three Bacillus thuringiensis subsp. israelensis d-endotoxins towards insect and mammalian cells, PLoS ONE, v.7, n.9, p.46121, 2012. Disponível em: <www.plosone.org/article>. Acesso em: 15 out. 2012.
• CRICKMORE, N.; ZEIGLER, D.R.; FEITELSON, J.; SCHNEPF, E.; VAN RIE, J.; LERECUS, D.; BAUM, J.; DEAN, D.H. Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal protein. Microbiology and Molecular Biology Reviews, v.62, n.3, p.807-813, 1998.
• CRICKMORE, N. Bacillus thuringiensis toxin nomenclature. 2012. Disponível em: <http://www.lifesci.sussex.ac.uk/home/Neil_CRICKMORE/Bt/> Acesso em: 15 out. 2012.
• FIUZA, L.M. Éstude des sites récepteurs et de la toxicité des d-endotoxines de Bacillus thuringiensis Berliner chez les larves de la Pyrale du riz Chilo supressalis Walker 1995. 160p. These (Doctorat) - Escole National Superieure Agronomique de Montpellier, França, 1995.
• GHAZALEH, F.A.; ARAÚJO, C.F.; FIDALGO, C.; CARLINI, C.R. Canatoxin induces activation on mice peritoneal macrophages. Brazilian Journal of Medical and Biological Research, v.25, p.1033-1035, 1992.
• GOHAR, M.; PERCHAT, S. Sample preparation for b-exotoxin determination in Bacillus thuringiensis cultures by reversed-phase high-performance liquid chromatography. Analitical Biochemistry, v.298, p.112-117, 2001.
• HERNÁNDEZ, C.S.; MARTÍNEZ, C.; PORCAR, M.; CABALLERO, P.; FERRÉ, J. Correlation between serovars of Bacillus thuringiensis and type I b-exotoxin production. Journal of Invertebrate Pathology, v.82, p.57-62. 2003.
• HÖFTE, H.; WHITHELEY, H.R. Insecticidal crystal proteins of Bacillus thuringiensis Microbiology and Molecular Biology Reviews, v.53, p.242-255, 1989.
• JAMES, C. 2011. Disponível em: <http://www.isaaa.org/kc> Acesso em: 15 out. 2012.
  » link
• KESHRI, G.; LAKSHMI, V.; SINGH, M.M. Pregnancy interceptiveactivity of Melia azedarach Linn. in adult female Sprague-Dawley rats. Contraception, v.68, p.3003-3006, 2003.
• LAEMMLI, U. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, v.227, p.680-685, 1970.
• LERECLUS, D.; AGAISSE, H.; GOMINET, M.; SALAMITOU, S.; SANCHIS, V. Identification of a gene that positively regulates transcription of the phosphatidylinositol-specific phospholipase C gene at the stationary phase. Journal of Bacteriology, v.178, p.2749-2756, 1996.
• MACKEDONSKI, V.V.; HADJIOLOV, A.A. Preferential in vivo inhibition of ribosomal ribonucleic acid synthesis in mouse liver by the exotoxin of Bacillus thuringiensis FEBS Letters, v.21, p.211-214, 1972.
• McCLINTOCK, J.T.; SCHAEFFER, C.R.; SJOBLAD, R.D. A comparative review of the mammalian toxicity of Bacillus thuringiensis based pesticides. Pesticide Science, v.45, p.95-105, 1995.
• MARRONI, N.; CASADEVALL, M.; PANÈS, J.; PIERA, C.; JOU, J.M.; PIQUE, J.M. Effects of chronic normovolemic anemia on gastric microcirculation and ethanol induced gastric damage in rats. Digest and Disease Science, v.39, n.4, p.751-757, 1994.
• MÉNDEZ, M.C.; ARAGÃO, M.; ELIAS, F.; RIET-CORREA, F.; JUAN, E.G. Experimetal intoxication by the leaves of Melia azedarach (Meliaceae) in cattle. Pesquisa Veterinária Brasileira, v.22, n.1, p.19-24, 2002.
• MORENO-FIERROS, L.; GARCÍA, N.; GUTIÉRREZ, R.; LÓPEZ-REVILA, R.; VASQUÉS-PADRÓN, R.I. Intranasal, rectal and intraperitoneal immunization with protoxin Cry 1Ac from Bacillus thuringiensis induces compartmentalized serum, intestinal, vaginal and pulmonary immune response in Balb/c mice. Microbes and Infection, v.2, p.885-890, 2000.
• O'CALLAGHAN, M.O.; GLARE, T.R.; BURGESS, E.O.J.; MALONE, L.A. Effects os plantds genetically modofied for insect resistance on nontarget organismos. Annual Review of Entomology, v.50, p.271-292, 2005.
• PERANI, M.; BISHOP, A.H.; VAID, A. Prevalence of b-exotoxin, diarrhoeal toxin and specific d-endotoxin in natural isolates of Bacillus thuringiensis FEMS Microbiology Letters, v.160, p.55-60, 1998.
• PINTO, L.M.N.; FIUZA, L.M. PCR and bioassays screening of Bacillus thuringiensis isolates from rice-fields os Rio Grande do Sul, specific to lepidopterans and coleopterans. Brazilian Journal of Microbiology, v.34, p.305-310, 2003.
• PRASAD, S.S.S.V.; SHETHNA, Y.I. Enhancement of immune response by the proteinaceous crystal of Bacillus thuringiensis Biochemistry and Biophisic Research Communications, v.62, p.517-521, 1975.
• RABINOVITCH, L.; CAVADOS, C.F.G.; LIMA, M.M. Bacillus entomopatogênicos. Dos Bacillus entomopatogênicos: o que se espera? Biotecnologia Ciência e Desenvolvimento, v.6, p.40-41, 1998.
• SCHENPF, E.; CRICKMORE, N.; VANRIE, J.; BAUM, J.; FEITELSON, J.; ZEIGLER, D.R.; DEAN, D.H. Bacillus thuringiensis and its pesticide crystal proteins. Microbiology and Molecular Biology Revision, v.62, p.775-806, 1998.
• SCHRODER, M.; POULSEN, M.; WILCKS, A.; KROGHSBO, S.; MILLER, A.; FRENZEL, T.; DANIER, J.; RYCHLIK, M.; EMAMI, K.; GATEHOUSE, A.; SHU, Q.; ENGEL, K.H.; ALTOSAAR, I.; KNUDSEN, I. A 90-day safety study of genetically modified rice expressing Cry1Ab protein (Bacillus thuringiensis toxin) in Wistar rats. Food and Chemical Toxicology, v.45, p.339-349, 2007.
• SEBESTA, K.; FARKAS, J.; HORSKA, K.; VAN KOVA, J. Thuringensin, the beta-exotoxin of Bacillus thuringiensis In: BURGES, H.D. Microbial control of pests and plant disesases 1970-80 London: Academic, 1981. p.249-281.
• SHARMA, C.B.S.R.; SAHU, R.K. A preliminary study on the responses of root meristems to exotoxin from Bacillus thuringiensis a constituent of a microbial insecticide, Thuricide. Mutation Research, v.46, p.19-26, 1977.
• SHELTON, A.M.; AHAO, J.Z.; ROUSH, R.T. Economic, ecologycal, food safety and social consequences of the deployment of Bt trangenic plants. Annual Review of Entomology, v.47, p.845-881, 2002.
• SIEGEL, J.P. Testing the pathogenicity and infectivity of entomopathogens to mammals. In: LACEY, L.A. (Ed.). Manual of techniques in insect pathology San Diego: Academic Press, 1997. p.325-336.
• SIEGEL, J.P. The mammalian safety of Bacillus thuringiensis based insecticides. Journal of Invertebrate Pathology, v.77, p.13-21, 2001.
• TIMM, C.D.; RIET-CORREA, F. Plantas tóxicas para suínos. Ciencia Rural, v.27, n.3, p.521-528, 1997.
• VÁSQUEZ-PADRÓN. R.I.; MORENO-FIERROS, L.; NERI-BAZAN, L.; MARTÍNEZ-GILL, A.F.; DE LA RIVA, G.A.; LOPÉZ-REVILLA, R. Characterization of the mucosal and systemic immune response induced by Cry 1Ac protein from Bacillus thruringiensis HD 73 in mice. Brazilian Journal of Medical and Biological Research, v.33, p.147-155, 2000.
• VIDAL, C.S.; ANDRADE, M.V.M.; VIANA, G. Ligadura do esôfago abdominal diminui a secreção gástrica induzida por toxina de escorpião em ratos. Acta Cirurgica Brasileira, v.19, n.3, p.166-175, 2004.
• VILAS-BÔAS, G.F.L.T.; VILAS-BÔAS, L.A.; LERECUS, D.; ARANTES, O.M.N. Bacillus thuringiensis conjugation under environmental conditions. FEMS Microbiology Ecology, v.25, p.369-374, 1998.
• VILAS-BÔAS, G.T.; ALVAREZ, R.C.; SANTOS, C.A.; VILAS-BOAS, L.A. Fatores de Virulência de Bacillus thuringiensis Berliner: O Que Existe Além das Proteínas Cry? EntomoBrasilis, v.5, n.1, p.1-10, 2012.
• WORLD HEALTH ORGANIZATION. Informal consultation on the development of Bacillus sphaericus as microbial larvicide. Geneva: UNDP;World Bank; WHO, 1985. 24p. (Special programmer for research and training in tropical diseases).
• YAMASHITA, S.; AKAO, T.; MIZUKI, E.; SAITOH, H.; HIGUCHI, K.; KIM, H.; OHBA, M. Characterization of the anti-cancer-cell paraesporal proteins of a Bacillus thuringiensis isolate. Canadian Journal of Microbiology, v.46, p.913-919, 2000.
• ZAHNER, V.; CABRAL, D.A.; RÉGUA-MANGIA, A.H.; RABINOVITCH, L.; MOREAU, G.; MCINTOSH, D. Distribuition of genes encoding putative virulence factors and fragment length polymorphisms in the vrrA gene among brazilian isolates of Bacillus cereus and Bacillus thuringiensis Applied Environmental Microbiology, v.71, n.12, p.8107-8114, 2005.

Publication Dates

• Publication in this collection
  03 June 2013
• Date of issue
  Dec 2012

History

• Received
  30 June 2011
• Accepted
  16 Oct 2012