RESUMEN
Castor cake is a by-product of the extraction of oil from from seeds of castor plants (Ricinus communis). This by-product contains high levels of proteins, but a toxic protein, ricin, limits its use as an animal feed. Ricin can be efficiently inactivated by treatment with calcium oxide (CaO), which can be evaluated by a cytotoxicity assay using LLC-MK2 cells. The mechanism by which the CaO treatment inactivates ricin, however, is unclear. We report the structural changes responsible for ricin inactivation. Purified ricin was treated with 0.6% CaO and then analyzed by mass spectrometry. This treatment degraded the ricin at preferential sites. The aqueous CaO solution had a pH >12, which preferentially cleaved asparagine residues, followed by glutamine, serine and glycine residues. The alkaline pH affected the tertiary structure of the ricin, cleaving its polypeptide chains and thereby eliminating its cytotoxic activity.
Asunto(s)
Citotoxinas/toxicidad , Ricina/toxicidad , Animales , Compuestos de Calcio/farmacología , Línea Celular , Óxidos/farmacología , Proteómica , Ricina/antagonistas & inhibidoresRESUMEN
Ricinus communis L. is of great economic importance due to the oil extracted from its seeds. Castor oil has been used for pharmaceutical and industrial applications, as a lubricant or coating agent, as a component of plastic products, as a fungicide or in the synthesis of biodiesel fuels. After oil extraction, a castor cake with a large amount of protein is obtained. However, this by-product cannot be used as animal feed due to the presence of toxic (ricin) and allergenic (2S albumin) proteins. Here, we propose two processes for detoxification and allergen inactivation of the castor cake. In addition, we establish a biological test to detect ricin and validate these detoxification processes. In this test, Vero cells were treated with ricin, and cell death was assessed by cell counting and measurement of lactate dehydrogenase activity. The limit of detection of the Vero cell assay was 10 ng/mL using a concentration of 1.6 x 10(5) cells/well. Solid-state fermentation (SSF) and treatment with calcium compounds were used as cake detoxification processes. For SSF, Aspergillus niger was grown using a castor cake as a substrate, and this cake was analyzed after 24, 48, 72, and 96 h of SSF. Ricin was eliminated after 24 h of SSF treatment. The cake was treated with 4 or 8% Ca(OH)2 or CaO, and both the toxicity and the allergenic properties were entirely abolished. A by-product free of toxicity and allergens was obtained.
Asunto(s)
Animales , Alérgenos/efectos de los fármacos , Aspergillus niger/crecimiento & desarrollo , Compuestos de Calcio/farmacología , Ricinus communis/efectos de los fármacos , Inactivación Metabólica , Alérgenos/toxicidad , Aspergillus niger/efectos de los fármacos , Chlorocebus aethiops , Ricinus communis/toxicidad , Muerte Celular/efectos de los fármacos , Degranulación de la Célula/efectos de los fármacos , Activación Enzimática , Fermentación , L-Lactato Deshidrogenasa/metabolismo , Mastocitos/efectos de los fármacos , Ricina/aislamiento & purificación , Ricina/toxicidad , Factores de Tiempo , Pruebas de Toxicidad , /aislamiento & purificación , /toxicidad , Células VeroRESUMEN
Ricinus communis L. is of great economic importance due to the oil extracted from its seeds. Castor oil has been used for pharmaceutical and industrial applications, as a lubricant or coating agent, as a component of plastic products, as a fungicide or in the synthesis of biodiesel fuels. After oil extraction, a castor cake with a large amount of protein is obtained. However, this by-product cannot be used as animal feed due to the presence of toxic (ricin) and allergenic (2S albumin) proteins. Here, we propose two processes for detoxification and allergen inactivation of the castor cake. In addition, we establish a biological test to detect ricin and validate these detoxification processes. In this test, Vero cells were treated with ricin, and cell death was assessed by cell counting and measurement of lactate dehydrogenase activity. The limit of detection of the Vero cell assay was 10 ng/mL using a concentration of 1.6 x 10(5) cells/well. Solid-state fermentation (SSF) and treatment with calcium compounds were used as cake detoxification processes. For SSF, Aspergillus niger was grown using a castor cake as a substrate, and this cake was analyzed after 24, 48, 72, and 96 h of SSF. Ricin was eliminated after 24 h of SSF treatment. The cake was treated with 4 or 8% Ca(OH)2 or CaO, and both the toxicity and the allergenic properties were entirely abolished. A by-product free of toxicity and allergens was obtained.
Asunto(s)
Alérgenos/efectos de los fármacos , Aspergillus niger/crecimiento & desarrollo , Compuestos de Calcio/farmacología , Inactivación Metabólica , Ricinus communis/efectos de los fármacos , Albuminas 2S de Plantas/aislamiento & purificación , Albuminas 2S de Plantas/toxicidad , Alérgenos/toxicidad , Animales , Aspergillus niger/efectos de los fármacos , Ricinus communis/toxicidad , Muerte Celular/efectos de los fármacos , Degranulación de la Célula/efectos de los fármacos , Chlorocebus aethiops , Activación Enzimática , Fermentación , L-Lactato Deshidrogenasa/metabolismo , Mastocitos/efectos de los fármacos , Ricina/aislamiento & purificación , Ricina/toxicidad , Factores de Tiempo , Pruebas de Toxicidad , Células VeroRESUMEN
Toxoplasma, which infects all eukaryotic cells, is considered to be a good system for the study of drug action and of the behavior of infected host cells. In the present study, we asked if thiosemicarbazone derivatives can be effective against tachyzoites and which morphological and ultrastructural features of host cells and parasites are associated with the destruction of Toxoplasma. The compounds were tested in infected Vero cell culture using concentration screens (0.1 to 20 mM). The final concentration of 1 mM was chosen for biological assay. The following results were obtained: 1) These new derivatives decreased T. gondii infection with an in vitro parasite IC50 percent of 0.2-0.7 mM, without a significant effect on host cells and the more efficient compounds were 2, 3 (thiosemicarbazone derivatives) and 4 (thiazolidinone derivative); 2) The main feature observed during parasite elimination was continuous morphological disorganization of the tachyzoite secretory system, progressive organelle vesiculation, and then complete disruption; 3) Ultrastructural assays also revealed that progressive vesiculation in the cytoplasm of treated parasites did not occur in the host cell; 4) Vesiculation inside the parasite resulted in death, but this feature occurred asynchronously in different intracellular tachyzoites; 5) The death and elimination of T. gondii was associated with features such as apoptosis-like stage, acidification and digestion of parasites into parasitophorous vacuoles. Our results suggest that these new chemical compounds are promising for the elimination of intracellular parasites by mainly affecting tachyzoite development at 1 mM concentration for 24 h of treatment.
Asunto(s)
Animales , Antiprotozoarios/farmacología , Tiazoles/farmacología , Tiosemicarbazonas/farmacología , Toxoplasma/efectos de los fármacos , Antiprotozoarios/química , Chlorocebus aethiops , Interacciones Huésped-Parásitos , Microscopía Electrónica de Transmisión , Pruebas de Sensibilidad Parasitaria , Tiazoles/química , Tiosemicarbazonas/química , Toxoplasma/ultraestructura , Células Vero/parasitologíaRESUMEN
Toxoplasma, which infects all eukaryotic cells, is considered to be a good system for the study of drug action and of the behavior of infected host cells. In the present study, we asked if thiosemicarbazone derivatives can be effective against tachyzoites and which morphological and ultrastructural features of host cells and parasites are associated with the destruction of Toxoplasma. The compounds were tested in infected Vero cell culture using concentration screens (0.1 to 20 mM). The final concentration of 1 mM was chosen for biological assay. The following results were obtained: 1) These new derivatives decreased T. gondii infection with an in vitro parasite IC50% of 0.2-0.7 mM, without a significant effect on host cells and the more efficient compounds were 2, 3 (thiosemicarbazone derivatives) and 4 (thiazolidinone derivative); 2) The main feature observed during parasite elimination was continuous morphological disorganization of the tachyzoite secretory system, progressive organelle vesiculation, and then complete disruption; 3) Ultrastructural assays also revealed that progressive vesiculation in the cytoplasm of treated parasites did not occur in the host cell; 4) Vesiculation inside the parasite resulted in death, but this feature occurred asynchronously in different intracellular tachyzoites; 5) The death and elimination of T. gondii was associated with features such as apoptosis-like stage, acidification and digestion of parasites into parasitophorous vacuoles. Our results suggest that these new chemical compounds are promising for the elimination of intracellular parasites by mainly affecting tachyzoite development at 1 mM concentration for 24 h of treatment.