RESUMO
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.
Assuntos
Citotoxinas/toxicidade , Ricina/toxicidade , Animais , Compostos de Cálcio/farmacologia , Linhagem Celular , Óxidos/farmacologia , Proteômica , Ricina/antagonistas & inibidoresRESUMO
Toxoplasma gondii is a parasite of great medical and veterinary importance that has worldwide distribution and causes toxoplasmosis. There are few treatments available for toxoplasmosis and the search for plant extracts and compounds with anti-Toxoplasma activity is of utmost importance for the discovery of new active drugs. The objective of this study was to investigate the action of a protein extract and a protease inhibitor enriched fraction from J. curcas seed cake on developing tachyzoites of T. gondii-infected Vero cells. The protein extract (JcCE) was obtained after solubilization of the J. curcas seed cake with 100 mM sodium borate buffer, pH 10, centrifugation and dialysis of the resulting supernatant with the extracting buffer. JcCE was used for the in vitro assays of anti-Toxoplasma activity at 0.01, 0.1, 0.5, 1.5, 3.0 and 5.0 mg/ml concentration for 24 h. The results showed that JcCE reduced the percentage of infection and the number of intracellular parasites, but had no effect on the morphology of Vero cells up to 3.0 mg/mL. The cysteine protease inhibitor enriched fraction, which was obtained after chromatography of JcCE on Sephadex G-75 and presented a unique protein band following SDS-PAGE, reduced both the number of T. gondii infected cells and intracellular parasites. These results suggest that both JcCE and the cysteine protease inhibitor enriched fraction interfere with the intracellular growth of T. gondii.
Assuntos
Antiprotozoários/farmacologia , Jatropha/química , Extratos Vegetais/farmacologia , Sementes/química , Toxoplasma/efeitos dos fármacos , Toxoplasmose/parasitologia , Animais , Antiprotozoários/química , Antiprotozoários/isolamento & purificação , Chlorocebus aethiops , Humanos , Masculino , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Inibidores de Proteases/química , Inibidores de Proteases/isolamento & purificação , Inibidores de Proteases/farmacologia , Toxoplasma/crescimento & desenvolvimento , Toxoplasmose/tratamento farmacológico , Células VeroRESUMO
The present study investigated the testis and sperm morphology of the tropical fish Gymnotus carapo after exposure to increasing CdCl2 concentrations (5-40 µM) for 24 and 96 h. The treatments induced Cd accumulation in the testis and a decrease in the gonadosomatic index from a 10 µM. Cd induced alterations in testis since 24h; however the extension and severity of damages increased after 96 h in all tested concentrations. Marked variations in the cysts size, proliferation of the interstitial tissue, infiltration of inflammatory cells, necrosis, reduction of germ cells and sperm aggregation was observed in 96 h treated fishes. In this time, there was a complete absence of germ cells in the testis of fish treated with 40 µM. The ultrastructural analysis allowed for the visualization of the initial damages over germ cells, such as the presence of vacuoles in the cytoplasm of spermatogonia, spermatocytes, and spermatids. Exposed fish (20 µM for 24 and 96 h) had alterations in sperm number and morphology. These results are important for establishing a direct correlation between the Cd accumulation and incidence of damages and can help characterize the mechanism of Cd-induced pathogenesis in the male reproductive system.
Assuntos
Cádmio/toxicidade , Peixes , Espermatócitos/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Testículo/efeitos dos fármacos , Animais , Evolução Biológica , Masculino , Espermátides/citologia , Espermátides/efeitos dos fármacos , Espermatócitos/citologia , Espermatogônias/citologia , Espermatogônias/efeitos dos fármacos , Espermatozoides/citologia , Testículo/citologiaRESUMO
This study investigated the progressive effects of HgCl2 in the testis and sperm of the tropical fish tuvira Gymnotus carapo L. exposed to increasing Hg concentrations (5-30 µm) and increasing exposure times (24-96 h). Histopathology and metal concentrations in the testis were observed. Hg concentrations in the testis reached 5.1 and 5.2 µg g(-1) in fish exposed to 20 and 30 µm of Hg, respectively. Hg effects on testicular tissue were observed even at low Hg concentrations, with no alterations in gonadosomatic index. However, the quantitative analysis of the induced alterations (lesion index) demonstrated that the Hg effects in testis became more severe after exposure to higher concentrations (20 and 30 µm) and during longer exposure (72 and 96 h), probably leading to partial or total loss of the organ function. Hg exposure (20 µm) also affected sperm count and altered sperm morphology. This study showed that HgCl2 caused progressive damage to testicular tissue, reduced sperm count and altered sperm morphology. These results are important in establishing a direct correlation between Hg accumulation and severity of lesions.
Assuntos
Gimnotiformes/metabolismo , Cloreto de Mercúrio/toxicidade , Espermatozoides/efeitos dos fármacos , Testículo/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Relação Dose-Resposta a Droga , Masculino , Microscopia Eletrônica de Varredura/veterinária , Espectrofotometria Atômica/veterinária , Espermatozoides/citologia , Fatores de TempoRESUMO
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.
Assuntos
Animais , Alérgenos/efeitos dos fármacos , Aspergillus niger/crescimento & desenvolvimento , Compostos de Cálcio/farmacologia , Ricinus communis/efeitos dos fármacos , Inativação Metabólica , Alérgenos/toxicidade , Aspergillus niger/efeitos dos fármacos , Chlorocebus aethiops , Ricinus communis/toxicidade , Morte Celular/efeitos dos fármacos , Degranulação Celular/efeitos dos fármacos , Ativação Enzimática , Fermentação , L-Lactato Desidrogenase/metabolismo , Mastócitos/efeitos dos fármacos , Ricina/isolamento & purificação , Ricina/toxicidade , Fatores de Tempo , Testes de Toxicidade , /isolamento & purificação , /toxicidade , Células VeroRESUMO
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.
Assuntos
Alérgenos/efeitos dos fármacos , Aspergillus niger/crescimento & desenvolvimento , Compostos de Cálcio/farmacologia , Inativação Metabólica , Ricinus communis/efeitos dos fármacos , Albuminas 2S de Plantas/isolamento & purificação , Albuminas 2S de Plantas/toxicidade , Alérgenos/toxicidade , Animais , Aspergillus niger/efeitos dos fármacos , Ricinus communis/toxicidade , Morte Celular/efeitos dos fármacos , Degranulação Celular/efeitos dos fármacos , Chlorocebus aethiops , Ativação Enzimática , Fermentação , L-Lactato Desidrogenase/metabolismo , Mastócitos/efeitos dos fármacos , Ricina/isolamento & purificação , Ricina/toxicidade , Fatores de Tempo , Testes de Toxicidade , Células VeroRESUMO
Contamination of c anine semen with urine can drastically reduce sperm motility. To assess whether this effect is associated with differences in pH and osmolarity, the present study evaluated canine semen diluted in prostatic fraction, autologous urine, ultrapure water and NaCl solutions containing the following osmolarities: 133, 260, 392, 519 and 860 mOsmol/ l . The semen dilutions were incubated f or 1 h at 37°C. Total and progressive sperm motility, membrane and acrosomal integrity and sperm morphology were evaluated. Semen and urine pH values were similar (semen = 6.5 ± 0.3; urine = 6.9 ± 0.3). After 1 h of incubation, total and progressive motility in the prostatic fraction were similar to that of sperm in the 260 mOsmol/ l solution (nearly 75% for total motility and 26 - 40% for progressive motility) and higher than observed in the other solutions, which had a total motility lower than 35% and a progressive motility under 3.2%. Compared to sperm in the seminal plasma, membrane integrity was lower in sperm incubated in water, solutions with 133 and 860 mOsmol/ l and urine (0 - 50% integrity), and acrosomal integrity was lower in sperm incubated in water, 860 mOsmol/ l solution and urine (0 - 32% integrity). Morphology changes (bent tail) were detected only in sperm incubated in the most hyposmotic solutions (water and 133 mOsmol/l ). In conclusion, pH is most likely not associated with the deleterious effects of urine on semen quality, and although only the most hyposmotic solutions caused morphological changes in sperm, sperm motility is compromised in solutions that are highly hypo - or hyperosmotic, such as urin.(AU)
Assuntos
Animais , Cães , Análise do Sêmen/veterinária , Sêmen/citologia , Concentração Osmolar , Urina , Cães/classificação , Contaminação Biológica/análiseRESUMO
Contamination of c anine semen with urine can drastically reduce sperm motility. To assess whether this effect is associated with differences in pH and osmolarity, the present study evaluated canine semen diluted in prostatic fraction, autologous urine, ultrapure water and NaCl solutions containing the following osmolarities: 133, 260, 392, 519 and 860 mOsmol/ l . The semen dilutions were incubated f or 1 h at 37°C. Total and progressive sperm motility, membrane and acrosomal integrity and sperm morphology were evaluated. Semen and urine pH values were similar (semen = 6.5 ± 0.3; urine = 6.9 ± 0.3). After 1 h of incubation, total and progressive motility in the prostatic fraction were similar to that of sperm in the 260 mOsmol/ l solution (nearly 75% for total motility and 26 - 40% for progressive motility) and higher than observed in the other solutions, which had a total motility lower than 35% and a progressive motility under 3.2%. Compared to sperm in the seminal plasma, membrane integrity was lower in sperm incubated in water, solutions with 133 and 860 mOsmol/ l and urine (0 - 50% integrity), and acrosomal integrity was lower in sperm incubated in water, 860 mOsmol/ l solution and urine (0 - 32% integrity). Morphology changes (bent tail) were detected only in sperm incubated in the most hyposmotic solutions (water and 133 mOsmol/l ). In conclusion, pH is most likely not associated with the deleterious effects of urine on semen quality, and although only the most hyposmotic solutions caused morphological changes in sperm, sperm motility is compromised in solutions that are highly hypo - or hyperosmotic, such as urin.
Assuntos
Animais , Cães , Análise do Sêmen/veterinária , Concentração Osmolar , Sêmen/citologia , Urina , Contaminação Biológica/análise , Cães/classificaçãoRESUMO
Melia azedarach (cinnamon) and Azadirachta indica (neem) have a variety of biologically active ingredients against virus, bacteria and protozoan parasites; however, little is known about their action on Toxoplasma gondii intracellular development. Toxoplasma gondii infects all eukaryotic cells, where it establishes and multiplies inside a modified vacuole called the parasitophorous vacuole until the cell ruptures, re-infecting other cells and establishing the infection. There are no efficient chemotherapies for the elimination of T. gondii, minimizing side effects. In this study, we performed in vitro assays with neem and cinnamon aqueous extracts against the intracellular development of T. gondii tachyzoites. After treatment with neem and cinnamon for 24 h, the percentage of infected cells and the number of intracellular parasites drastically decreased. This effect was concentration-dependent. During the incubation of the extracts, progressive morphological and ultrastructure alterations led to intense vesiculation and complete elimination of the parasite from the intracellular medium. However, during the treatment with extracts, no morphological effects were observed in the structure of the host cell. These results suggest that the aqueous extracts of neem and cinnamon were capable of interfering with and eliminating the intracellular development of Toxoplasma gondii.
Melia azedarach (canela) e Azadirachta indica (nim) apresenta grande variedade de ingredientes biologicamente ativos contra vírus, bactérias e protozoários, mas nenhum efeito sobre o desenvolvimento intracelular do Toxoplasma gondii é conhecido. Toxoplasma gondii infecta todos os tipos de células Eucarióticas, onde se estabelece no meio intracelular em vacúolo modificado conhecido como vacúolo parasitóforo. Neste vacúolo ocorre a replicação levando a ruptura da célula hospedeira e reinfecção de novas células, perpetuando a infecção. A quimioterapia utilizada não é capaz de eliminar o parasita além de induzir fortes efeitos colaterais. Neste estudo, nós demonstramos o efeito in vitro de extratos aquosos da canela e nim sobre o desenvolvimento intracelular do taquizoíto do Toxoplasma gondii. Após tratamento de nim e canela por 24 h, a porcentagem de infecção e o número de taquizoítos intracelulares decaiu drasticamente. Este efeito foi concentração-dependente. Durante incubação dos extratos, uma progressiva desorganização morfológica e ultraestrutural levaram a formação de intensa vesiculação e completa destruição do parasita, que passou a uma estrutura amorfa, antes da completa eliminação do meio intracelular. No entanto durante o tratamento com os extratos, efeitos morfológicos não foram observados nas estruturas da célula hospedeira. Estes resultados sugerem que os extratos aquosos de nim e canela foram capazes de interferir e eliminar o desenvolvimento intracelular do Toxoplasma gondii.
Assuntos
Azadirachta/análise , Espaço Intracelular/parasitologia , Extratos Vegetais/química , Folhas de Planta/parasitologia , Técnicas In Vitro , Toxoplasma/parasitologia , Azadirachta/parasitologia , Cinnamomum zeylanicum/parasitologia , Citotoxinas/fisiologia , Citotoxinas/químicaRESUMO
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.