Full confluency, serum starvation, and roscovitine for inducing arrest in the G0/G1 phase of the cell cycle in puma skin-derived fibroblast lines

The puma population is constantly decreasing, and cloning by somatic cell nuclear transfer can be used to conserve the species. One of the factors determining the success of the development of cloned embryos is the cell cycle stage of the donor cells. We evaluated the effects of full confluency (~100%), serum starvation (0.5% serum), and roscovitine (15 µM) treatments on the cell cycle synchronization in G0/G1 of puma skin-derived fibroblasts by flow cytometric analysis. Also, we assessed the effects of these synchronization methods on morphology, viability, and apoptosis levels using microscopy tools. The results showed that culturing the cells to confluence for 24 h (84.0%), 48 h (84.6%), and 72 h (84.2%) and serum starvation for 96 h (85.4%) yielded a significantly higher percentage of cells arrested in the G0/G1 (P < 0.05) phase than cells not subjected to any cell cycle synchronization method (73.9%). Nevertheless, while serum starvation reduced the percentage of viable cells, no difference was observed for the full confluence and roscovitine treatments (P > 0.05). Moreover, roscovitine for 12 h (78.6%) and 24 h (82.1%) was unable to synchronize cells in G0/G1 (P > 0.05). In summary, full confluency induces puma fibroblast cell cycle synchronization at the G0/G1 stage without affecting cell viability. These outcomes may be valuable for planning donor cells for somatic cell nuclear transfer in pumas.(AU)

In vitro cytotoxicity of curcuminoids against head and neck cancer HNO97 cell line / Citotoxicidade in vitro de curcuminoides contra a linhagem de células HNO97 de câncer de cabeça e pescoço

Oral squamous cell carcinoma (OSCC) is a malignant tumour of Head and Neck Cancer (HNC). The recent therapeutic approaches used to treat cancer have adverse side effects. The natural agents exhibiting anticancer activities are generally considered to have a robust therapeutic potential. Curcuminoids, one of the major active compounds of the turmeric herb, are used as a therapeutic agent for several diseases including cancer. In this study, the cytotoxicity of curcuminoids was investigated against OSCC cell line HNO97. Our data showed that curcuminoids significantly inhibits the proliferation of HNO97 in a time and dose-dependent manner (IC50=35 μM). Cell cycle analysis demonstrated that curcuminoids increased the percentage of G2/M phase cell populations in the treated groups. Treating HNO97 cells with curcuminoids led to cell shrinking and increased detached cells, which are the typical appearance of apoptotic cells. Moreover, flow cytometry analysis revealed that curcuminoids significantly induced apoptosis in a time-dependent manner. Furthermore, as a response to curcuminoids treatment, comet tails were formed in cell nuclei due to the induction of DNA damage. Curcuminoids treatment reduced the colony formation capacity of HNO97 cells and induced morphological changes. Overall, these findings demonstrate that curcuminoids can in vitro inhibit HNC proliferation and metastasis and induce apoptosis.

O carcinoma de células escamosas oral (OSCC) é um tumor maligno do câncer de cabeça e pescoço (HNC). As recentes abordagens terapêuticas usadas para tratar o câncer têm efeitos colaterais adversos. Os agentes naturais que exibem atividades anticâncer são geralmente considerados como tendo um potencial terapêutico robusto. Curcuminoides, um dos principais compostos ativos da erva cúrcuma, são usados como agente terapêutico para várias doenças, incluindo câncer. Neste estudo, a citotoxicidade dos curcuminoides foi investigada contra a linha de células OSCC HNO97. Nossos dados mostraram que os curcuminoides inibem significativamente a proliferação de HNO97 de forma dependente do tempo e da dose (IC50 = 35 μM). A análise do ciclo celular demonstrou que os curcuminoides aumentaram a porcentagem de populações de células da fase G2 / M nos grupos tratados. O tratamento das células HNO97 com curcuminoides levou ao encolhimento celular e ao aumento das células destacadas, que são a aparência típica das células apoptóticas. Além disso, a análise de citometria de fluxo revelou que os curcuminoides induziram significativamente a apoptose de uma maneira dependente do tempo. Além disso, em resposta ao tratamento com curcuminoides, caudas de cometa foram formadas nos núcleos das células devido à indução de danos ao DNA. O tratamento com curcuminoides reduziu a capacidade de formação de colônias das células HNO97 e induziu alterações morfológicas. No geral, esses achados demonstram que os curcuminoides podem inibir in vitro a proliferação e metástase de HNC e induzir apoptose.

In vitro cytotoxicity of curcuminoids against head and neck cancer HNO97 cell line

Abstract Oral squamous cell carcinoma (OSCC) is a malignant tumour of Head and Neck Cancer (HNC). The recent therapeutic approaches used to treat cancer have adverse side effects. The natural agents exhibiting anticancer activities are generally considered to have a robust therapeutic potential. Curcuminoids, one of the major active compounds of the turmeric herb, are used as a therapeutic agent for several diseases including cancer. In this study, the cytotoxicity of curcuminoids was investigated against OSCC cell line HNO97. Our data showed that curcuminoids significantly inhibits the proliferation of HNO97 in a time and dose-dependent manner (IC50=35 M). Cell cycle analysis demonstrated that curcuminoids increased the percentage of G2/M phase cell populations in the treated groups. Treating HNO97 cells with curcuminoids led to cell shrinking and increased detached cells, which are the typical appearance of apoptotic cells. Moreover, flow cytometry analysis revealed that curcuminoids significantly induced apoptosis in a time-dependent manner. Furthermore, as a response to curcuminoids treatment, comet tails were formed in cell nuclei due to the induction of DNA damage. Curcuminoids treatment reduced the colony formation capacity of HNO97 cells and induced morphological changes. Overall, these findings demonstrate that curcuminoids can in vitro inhibit HNC proliferation and metastasis and induce apoptosis.

Resumo O carcinoma de células escamosas oral (OSCC) é um tumor maligno do câncer de cabeça e pescoço (HNC). As recentes abordagens terapêuticas usadas para tratar o câncer têm efeitos colaterais adversos. Os agentes naturais que exibem atividades anticâncer são geralmente considerados como tendo um potencial terapêutico robusto. Curcuminoides, um dos principais compostos ativos da erva cúrcuma, são usados como agente terapêutico para várias doenças, incluindo câncer. Neste estudo, a citotoxicidade dos curcuminoides foi investigada contra a linha de células OSCC HNO97. Nossos dados mostraram que os curcuminoides inibem significativamente a proliferação de HNO97 de forma dependente do tempo e da dose (IC50 = 35 M). A análise do ciclo celular demonstrou que os curcuminoides aumentaram a porcentagem de populações de células da fase G2 / M nos grupos tratados. O tratamento das células HNO97 com curcuminoides levou ao encolhimento celular e ao aumento das células destacadas, que são a aparência típica das células apoptóticas. Além disso, a análise de citometria de fluxo revelou que os curcuminoides induziram significativamente a apoptose de uma maneira dependente do tempo. Além disso, em resposta ao tratamento com curcuminoides, caudas de cometa foram formadas nos núcleos das células devido à indução de danos ao DNA. O tratamento com curcuminoides reduziu a capacidade de formação de colônias das células HNO97 e induziu alterações morfológicas. No geral, esses achados demonstram que os curcuminoides podem inibir in vitro a proliferação e metástase de HNC e induzir apoptose.

In vitro cytotoxicity of curcuminoids against head and neck cancer HNO97 cell line / Citotoxicidade in vitro de curcuminoides contra a linhagem de células HNO97 de câncer de cabeça e pescoço

Oral squamous cell carcinoma (OSCC) is a malignant tumour of Head and Neck Cancer (HNC). The recent therapeutic approaches used to treat cancer have adverse side effects. The natural agents exhibiting anticancer activities are generally considered to have a robust therapeutic potential. Curcuminoids, one of the major active compounds of the turmeric herb, are used as a therapeutic agent for several diseases including cancer. In this study, the cytotoxicity of curcuminoids was investigated against OSCC cell line HNO97. Our data showed that curcuminoids significantly inhibits the proliferation of HNO97 in a time and dose-dependent manner (IC50=35 μM). Cell cycle analysis demonstrated that curcuminoids increased the percentage of G2/M phase cell populations in the treated groups. Treating HNO97 cells with curcuminoids led to cell shrinking and increased detached cells, which are the typical appearance of apoptotic cells. Moreover, flow cytometry analysis revealed that curcuminoids significantly induced apoptosis in a time-dependent manner. Furthermore, as a response to curcuminoids treatment, comet tails were formed in cell nuclei due to the induction of DNA damage. Curcuminoids treatment reduced the colony formation capacity of HNO97 cells and induced morphological changes. Overall, these findings demonstrate that curcuminoids can in vitro inhibit HNC proliferation and metastasis and induce apoptosis.(AU)

O carcinoma de células escamosas oral (OSCC) é um tumor maligno do câncer de cabeça e pescoço (HNC). As recentes abordagens terapêuticas usadas para tratar o câncer têm efeitos colaterais adversos. Os agentes naturais que exibem atividades anticâncer são geralmente considerados como tendo um potencial terapêutico robusto. Curcuminoides, um dos principais compostos ativos da erva cúrcuma, são usados como agente terapêutico para várias doenças, incluindo câncer. Neste estudo, a citotoxicidade dos curcuminoides foi investigada contra a linha de células OSCC HNO97. Nossos dados mostraram que os curcuminoides inibem significativamente a proliferação de HNO97 de forma dependente do tempo e da dose (IC50 = 35 μM). A análise do ciclo celular demonstrou que os curcuminoides aumentaram a porcentagem de populações de células da fase G2 / M nos grupos tratados. O tratamento das células HNO97 com curcuminoides levou ao encolhimento celular e ao aumento das células destacadas, que são a aparência típica das células apoptóticas. Além disso, a análise de citometria de fluxo revelou que os curcuminoides induziram significativamente a apoptose de uma maneira dependente do tempo. Além disso, em resposta ao tratamento com curcuminoides, caudas de cometa foram formadas nos núcleos das células devido à indução de danos ao DNA. O tratamento com curcuminoides reduziu a capacidade de formação de colônias das células HNO97 e induziu alterações morfológicas. No geral, esses achados demonstram que os curcuminoides podem inibir in vitro a proliferação e metástase de HNC e induzir apoptose.(AU)

Effects of ß-glucan from S. cerevisiae on the expression of Casp9, Ccna2 and Sod1 genes in MCF-7 cells

ß-Glucans (ßG) are polysaccharides widely distributed in nature with chemopreventive properties. The aim of this study was to investigate the effects of ßG and the combined treatment with doxorubicin (Dox) on cell viability and mRNA levels of genes involved in cell cycle, apoptosis and antioxidant response. ßG was not cytotoxic. The mRNA levels of CCNA2of cells exposed to ß-glucan was upregulated and the exposure to Dox decreased the expression, while the combination led to an upregulation. Modulation of mRNA levels of CASP9suggest that ßG could inhibit promotion and progression steps of carcinogenesis, eliminatingneoplastic cells. The upregulation of CCNA2gene in combined treatment could be occurred due to ability of ßG in restoring the cell cycle distribution pattern after treatment with Dox. The upregulation of SOD1suggests that ßG can enhance the intracellular antioxidant defense, reducing the levels of superoxide dismutase induced by Dox. This response could reduce oxidative damage and attenuate tissue damage during chemotherapeutic treatment. Our data suggest that the drug combination may be less effective in killing tumor cells than the treatment with Dox alone. Thus, future studies should carefully consider this effect on indication of ßG during chemotherapy.Keywords:caspase-9; cyclin A2; superoxide dismutase 1; cell cycle; antioxidant.Received on July 2, 2020.Accepted on February 7, 2022.IntroductionGlucans are polysaccharides widely distributed in nature and oftenstudied due to chemopreventive properties. They are constituent of the cell wall of plants (oats and barley), algae, bacteria and fungi. ß-glucans (ßG)have a common structure comprising a main chain of ß-(1,3) and/or ß-(1,4) D-glucopyranosyl unit and they differ in length and branching structures. ßG of Saccharomyces cerevisiaehave 1→6 side branches while those of bacteria have 1→4 side branches (Chan, Chan, & Sze, 2009). ßGcan prevent DNA damage induced by chemical and physical agents (Ghavami,Goliaei, Taghizadeh, & Nikoofar, 2014). Some authors showed its significant efficacy in preventing mutagenic effects caused by doxorubicin, cyclophosphamide and cisplatin (Tohamy, El-Ghor, El-Nahas, & Noshy, 2003), methyl methanesulfonate (Oliveira et al., 2007)and hydrogen peroxide (Slamenová, 2003). Moreover, some studies have related the antioxidant ability of ßGagainst reactive free radicals formed by endogenous metabolic processes or exogenous chemicals (Tsiapali et al., 2001; Slamenová,2003; Sener, Eksioglu-Demiralp, Cetiner, Ercan, & Yegen, 2006; Guerra Dore et al., 2007; Kofuji et al., 2012; Lei et al., 2015). Yeast-derived ßGhave modulating action of humoral and cellular immune responses (Vetvicka et al., 2007).This activity provides protection to the organism against infections and cancer development (Samuelsen, Schrezenmeir, & Knutsen, 2014; Roudbary, Daneshmand, Hajimorad, Roudbarmohammadip, & Hassan, 2015). Despite postulated modes of action by which ß-glucan works are lacking information about the molecular mechanisms involved in the chemopreventive activity of this polysaccharide. In addition, compounds with chemopreventive properties can contribute to reduce side effects and toxicity during the chemotherapeutic treatment. Therefore, the aim of this study was to investigate the effects of ßG and the combined treatment with doxorubicin (Dox) on the expression of genes related with apoptosis (CASP9), cell cycle control (CCNA2)and antioxidant defense (SOD1)in human breast cancer MCF-7 cells. Doxorubicin (Dox) was chosen because it is one of the most used chemotherapeutic agent for cancer treatment. The limitation on the use of Dox in cancer treatment is the lack of selectivity against cancer cells and, consequently, its toxicity to patients.(AU)

Perfil proteico de oócitos caninos / Protein profile of canine oocytes

O presente estudo foi conduzido para caracterizaro proteoma de oócitos caninos. Oócitos foram coletados de 120 cadelas e apenas os COCs grau 1 foram selecionados para o cultivo in vitro. Após o cultivo, os oócitos foram submetidos à extração de proteínas. As proteínas foram digeridas com tripsina e analisadas por espectrometria de massa. Trinta e quatro proteínas foram identificadas nos oócitos caninos. Estas proteínas foram agrupadas em três categorias de acordo com a sua função biológica, molecular e localização celular. Quanto ao processo biológico, foram encontradas diversas proteínas envolvidas no ciclo celular, fertilização, regulação da transcrição e via de sinalização. A análise da ontologia do gene revelou alta porcentagem de proteínas envolvidas na atividade de ligação. Com base na análise da rede proteína-proteína usando a plataforma STRING, observou-se que a vimentina apresentou interações com as CASP3, CASP6, CASP7 e CASP8, envolvidos na apoptose. O componente de complemento C3, interagiu com receptores do complemento, como CR1 e CR2. A proteína de ligação retinol 4 interagiu com precursores de retinol. Actina esteve intimamente relacionada com as proteínas cofilinas 1e 2. A queratina 10 interagiu com a proteína CDK9 relacionada ao processo de sinalização celular. Essas proteínas são essenciais para o desenvolvimento completo de oócitos e fertilização. O presente estudo contém a primeira descrição da composição proteica dos oócitos caninos. A construção de bibliotecas de proteínas de oócitos, para cada espécie, estabelecerá as bases para a compreensão e o mapeamento dos eventos cruciais que definem a competência dos oócitos.

The present study was conducted to characterize the major proteome of canine oocytes. Ovaries were collected from 120 bitches and only Grade 1 COCs were selected for in vitro culture. After in vitro maturation, oocytes were subjected to protein extraction. Proteins were then trypsin-digested and analyzed by tandem mass spectrometry. Thirty-four proteins were identified in the canine oocytes. These proteins have been grouped into three different categories according to their biological, molecular function and cellular localization. With regard to biological process, we found many proteins involved in cell cycle, fertilization, transcription regulation and signaling pathway. The gene ontology analysis also revealeda high percentage of proteins involved in binding activity. Based on protein–protein network analysis using STRING platform, we found that vimentin presents links with CASP3, CASP6, CASP7 and CASP8, which are involved in apoptosis. Complement component C3, interacted with complement receptors, such as CR1 and CR2. Retinol-binding protein 4 interacted with retinol precursors. Actin potentially interacted with cofil in protein 1 and 2. Keratin 10, in turn, had interacted with CDK9, which are involved in pathway signaling. These proteins are essentials for the complete oocyte development and fertilization. In summary, the present study contains the first description of the main protein composition of canine oocytes. Construction of libraries of oocyte proteins, for each especies, will set the foundations for understanding and mapping the crucial events that define oocyte competence.

Perfil proteico de oócitos caninos / Protein profile of canine oocytes

O presente estudo foi conduzido para caracterizaro proteoma de oócitos caninos. Oócitos foram coletados de 120 cadelas e apenas os COCs grau 1 foram selecionados para o cultivo in vitro. Após o cultivo, os oócitos foram submetidos à extração de proteínas. As proteínas foram digeridas com tripsina e analisadas por espectrometria de massa. Trinta e quatro proteínas foram identificadas nos oócitos caninos. Estas proteínas foram agrupadas em três categorias de acordo com a sua função biológica, molecular e localização celular. Quanto ao processo biológico, foram encontradas diversas proteínas envolvidas no ciclo celular, fertilização, regulação da transcrição e via de sinalização. A análise da ontologia do gene revelou alta porcentagem de proteínas envolvidas na atividade de ligação. Com base na análise da rede proteína-proteína usando a plataforma STRING, observou-se que a vimentina apresentou interações com as CASP3, CASP6, CASP7 e CASP8, envolvidos na apoptose. O componente de complemento C3, interagiu com receptores do complemento, como CR1 e CR2. A proteína de ligação retinol 4 interagiu com precursores de retinol. Actina esteve intimamente relacionada com as proteínas cofilinas 1e 2. A queratina 10 interagiu com a proteína CDK9 relacionada ao processo de sinalização celular. Essas proteínas são essenciais para o desenvolvimento completo de oócitos e fertilização. O presente estudo contém a primeira descrição da composição proteica dos oócitos caninos. A construção de bibliotecas de proteínas de oócitos, para cada espécie, estabelecerá as bases para a compreensão e o mapeamento dos eventos cruciais que definem a competência dos oócitos.(AU)

The present study was conducted to characterize the major proteome of canine oocytes. Ovaries were collected from 120 bitches and only Grade 1 COCs were selected for in vitro culture. After in vitro maturation, oocytes were subjected to protein extraction. Proteins were then trypsin-digested and analyzed by tandem mass spectrometry. Thirty-four proteins were identified in the canine oocytes. These proteins have been grouped into three different categories according to their biological, molecular function and cellular localization. With regard to biological process, we found many proteins involved in cell cycle, fertilization, transcription regulation and signaling pathway. The gene ontology analysis also revealeda high percentage of proteins involved in binding activity. Based on protein–protein network analysis using STRING platform, we found that vimentin presents links with CASP3, CASP6, CASP7 and CASP8, which are involved in apoptosis. Complement component C3, interacted with complement receptors, such as CR1 and CR2. Retinol-binding protein 4 interacted with retinol precursors. Actin potentially interacted with cofil in protein 1 and 2. Keratin 10, in turn, had interacted with CDK9, which are involved in pathway signaling. These proteins are essentials for the complete oocyte development and fertilization. In summary, the present study contains the first description of the main protein composition of canine oocytes. Construction of libraries of oocyte proteins, for each especies, will set the foundations for understanding and mapping the crucial events that define oocyte competence.(AU)

Beta Lapachone blocks the cell cycle and induces apoptosis in canine osteosarcoma cells / Beta Lapachona bloqueia o ciclo celular e induz apoptose em células de osteossarcoma canino

Osteosarcoma is a malignant tumor of primitive bone cells with a high incidence in dogs and humans. The need for more effective drugs with less adverse consequences has pushed the development of chemotherapeutic agents from plants and other natural sources. The aim of this study was to verify the cytotoxic effects of beta-lapachone, a compound present in the sawdust of Tabebuia sp. (popularly known as ipê) wood, on canine osteosarcoma cells subcultured and treated in different concentrations (0.1µm, 0.3µm e 1.0µm) and exposure times (24h, 48h e 72h). Results were obtained through Trypan blue dye exclusion, tetrazolium reducing method, cell survival assay, Annexin V-FITC and Propidium Iodine labeling, JC-1 dye labeling and cell cycle kinetics e analysis. The group treated with 0.3µm beta-lapachone presented higher decrease in cell viability (80.27%, 24h, 47.41%, 48h and 35.19%, 72h) and greater progression of cytotoxicity (19.73%, 24h, 52.59%, 48h and 64.81%, 72h). The lower IC50 (0.180µm) was verified in the group treated for 72 hours. Cell growth after treatment decreased as concentration and time of exposure increased, with 0.50% survival fraction at the concentration of 1.0µm. Initial apoptosis was the most frequent type of cell death in all groups, reaching bottom in the 24-hour group treated with 0.1µm (4.26%) and peaking in the 72-hour group treated with 1.0µm (85.89%). Mitochondrial depolarization demonstrated a dose-dependent phenomenon, indicating the intrinsic apoptosis. Cell growth inhibition by blocking cell cycle in the G0/G1 phase related to the exposure the time. β-lapachone is cytotoxic for canine osteosarcoma cells, induces apoptosis and promotes cell cycle arrest in G0/G1 phase.(AU)

O osteossarcoma é o tumor maligno das células ósseas primitivas, com alta incidência em cães e humanos. A necessidade de medicamentos mais efetivos, com menor consequência adversa, tem gerado esforços para o desenvolvimento de agentes quimioterápicos compostos por plantas e outras fontes naturais. O objetivo deste estudo foi verificar os efeitos citotóxicos da beta lapachona, um composto presente na serragem da madeira do ipê, sobre células de osteossarcoma canino subcultivadas e submetidas ao tratamento, de acordo com as diferentes concentrações (0.1µm, 0.3µm e 1.0µm) e tempo de exposição (24h, 48h e 72h). Os resultados foram obtidos por meio dos métodos de exclusão do corante azul de Tripan e de redução do tetrazólio, além dos ensaios de sobrevivência celular, de dupla marcação com Anexina V-FITC e Iodeto de Propídio, de marcação com o corante JC-1 e pela análise da cinética do ciclo celular. O grupo tratado com 0.3µm de beta lapachona apresentou melhor regressão da viabilidade celular (80,27%, 24h; 47,41%, 48h e 35,19%, 72h) e maior progressão da citotoxicidade (19,73%, 24h; 52,59%, 48h e 64,81%, 72h). O menor IC50 (0.180µm) ocorreu no grupo tratado por 72 horas. O crescimento celular após o tratamento foi menor, de acordo com o aumento da concentração e tempo de exposição, apresentando 0,50% de fração de sobrevivência na concentração de 1.0µm. A apoptose inicial foi o tipo de morte celular mais frequente em todos os grupos, menor no grupo de 24 horas tratado com 0.1µm (4,26%) e maior no grupo de 72 horas tratado com 1.0µm (85,89%). A despolarização mitocondrial ocorreu de maneira dose dependente, indicando a ocorrência de apoptose intrínseca. A β lapachona possui efeitos citotóxicos em células de osteossarcoma canino, induz apoptose e promove o bloqueio do ciclo celular na fase G0/G1.(AU)

Beta Lapachone blocks the cell cycle and induces apoptosis in canine osteosarcoma cells / Beta Lapachona bloqueia o ciclo celular e induz apoptose em células de osteossarcoma canino

Osteosarcoma is a malignant tumor of primitive bone cells with a high incidence in dogs and humans. The need for more effective drugs with less adverse consequences has pushed the development of chemotherapeutic agents from plants and other natural sources. The aim of this study was to verify the cytotoxic effects of beta-lapachone, a compound present in the sawdust of Tabebuia sp. (popularly known as ipê) wood, on canine osteosarcoma cells subcultured and treated in different concentrations (0.1µm, 0.3µm e 1.0µm) and exposure times (24h, 48h e 72h). Results were obtained through Trypan blue dye exclusion, tetrazolium reducing method, cell survival assay, Annexin V-FITC and Propidium Iodine labeling, JC-1 dye labeling and cell cycle kinetics e analysis. The group treated with 0.3µm beta-lapachone presented higher decrease in cell viability (80.27%, 24h, 47.41%, 48h and 35.19%, 72h) and greater progression of cytotoxicity (19.73%, 24h, 52.59%, 48h and 64.81%, 72h). The lower IC50 (0.180µm) was verified in the group treated for 72 hours. Cell growth after treatment decreased as concentration and time of exposure increased, with 0.50% survival fraction at the concentration of 1.0µm. Initial apoptosis was the most frequent type of cell death in all groups, reaching bottom in the 24-hour group treated with 0.1µm (4.26%) and peaking in the 72-hour group treated with 1.0µm (85.89%). Mitochondrial depolarization demonstrated a dose-dependent phenomenon, indicating the intrinsic apoptosis. Cell growth inhibition by blocking cell cycle in the G0/G1 phase related to the exposure the time. β-lapachone is cytotoxic for canine osteosarcoma cells, induces apoptosis and promotes cell cycle arrest in G0/G1 phase.(AU)

O osteossarcoma é o tumor maligno das células ósseas primitivas, com alta incidência em cães e humanos. A necessidade de medicamentos mais efetivos, com menor consequência adversa, tem gerado esforços para o desenvolvimento de agentes quimioterápicos compostos por plantas e outras fontes naturais. O objetivo deste estudo foi verificar os efeitos citotóxicos da beta lapachona, um composto presente na serragem da madeira do ipê, sobre células de osteossarcoma canino subcultivadas e submetidas ao tratamento, de acordo com as diferentes concentrações (0.1µm, 0.3µm e 1.0µm) e tempo de exposição (24h, 48h e 72h). Os resultados foram obtidos por meio dos métodos de exclusão do corante azul de Tripan e de redução do tetrazólio, além dos ensaios de sobrevivência celular, de dupla marcação com Anexina V-FITC e Iodeto de Propídio, de marcação com o corante JC-1 e pela análise da cinética do ciclo celular. O grupo tratado com 0.3µm de beta lapachona apresentou melhor regressão da viabilidade celular (80,27%, 24h; 47,41%, 48h e 35,19%, 72h) e maior progressão da citotoxicidade (19,73%, 24h; 52,59%, 48h e 64,81%, 72h). O menor IC50 (0.180µm) ocorreu no grupo tratado por 72 horas. O crescimento celular após o tratamento foi menor, de acordo com o aumento da concentração e tempo de exposição, apresentando 0,50% de fração de sobrevivência na concentração de 1.0µm. A apoptose inicial foi o tipo de morte celular mais frequente em todos os grupos, menor no grupo de 24 horas tratado com 0.1µm (4,26%) e maior no grupo de 72 horas tratado com 1.0µm (85,89%). A despolarização mitocondrial ocorreu de maneira dose dependente, indicando a ocorrência de apoptose intrínseca. A β lapachona possui efeitos citotóxicos em células de osteossarcoma canino, induz apoptose e promove o bloqueio do ciclo celular na fase G0/G1.(AU)

Expression dynamics of caveolin-1 in fibroblasts of newborn rats with chronic lung disease and its impact on lung fibroblast proliferation

Purpose: To evaluate the changes of caveolin-1 in lung fibroblasts in newborn Wistar rats when exposed to hyperoxic conditions, as well as lung fibroblasts cell cycle. Methods: One hundred newborn Wistar rats were randomly divided (50 rats/group) into experimental and control groups, exposed to hyperoxic conditions or normal air, respectively. The fraction of inspired oxygen (FiO2) in the experimental group was 90%, whereas this value was 21% in the control group. Lung fibroblasts were collected on days 3, 7, and 14 of the experiment. Caveolin-1 expression dynamics in lung fibroblasts was assayed in each group by immunofluorescence and Western blot analyses. Flow cytometry (FCM) was used to assess the proportions of lung fibroblasts at different stages of the cell cycle. Results: On day 3, no significant difference in caveolin-1 expression was observed between the hyperoxic and control groups; however, on days 7 and 14, caveolin-1 expression was significantly lower in the hyperoxic group than in the control (P 0.05). No apparent differences were observed in caveolin-1 expression in the control group at the different time points. Using FCM analysis, we showed that the proportion of lung fibroblasts in G0/G1 phase in the hyperoxic group decreased compared to that of the control group on day 7, while the proportion of S-phase cells increased (P 0.05). These differences were more significant when the groups were compared on day 14 (P 0.01). Conclusion: After seven days the exposure to hyperoxic conditions, lung fibroblasts proliferated and caveolin-1 expression decreased.(AU)

Antiproliferative and genotoxic effects of nature identical and artificial synthetic food additives of aroma and flavor / Efeito antiproliferativo e genotóxico de aditivos alimentares de aroma e sabor sintéticos, dos tipos idêntico ao natural e artificial

This study aimed to analyze the antiproliferative and genotoxic potential of synthetic food flavorings, nature identical passion fruit and artificial vanilla. This assessment used root meristem cells of Allium cepa L., in exposure times of 24 and 48 hours and using doses of 0.2; 0.4 and 0.6 mL. Roots were fixed in Carnoys solution, hydrolyzed in hydrochloric acid, stained with acetic orcein and analyzed with optical microscope at 400× magnification, 5,000 cells for each treatment. For data analysis, it was used Chi-square test at 5%. Doses of 0.2 mL at ET 48 h; 0.4 and 0.6 mL at ET 24 and 48 h of passion fruit flavor, and the three doses of the vanilla flavor at ET 24 and 48 h significantly reduced the cell division rate in the meristems of roots, proving to be cytotoxic. Doses of 0.2; 0.4 and 0.6 mL of the passion fruit additive, and the three doses of vanilla tested, in the two exposure times, induced mitotic spindle changes and micronuclei formation in the cells of the test organism used, proving to be genotoxic. Therefore, under the studied conditions, flavoring solutions of vanilla and passion fruit, marketed nationally and internationally, significantly altered the functioning of the cell cycle in root meristem cells of A. cepa.(AU)

Neste trabalho teve-se por objetivo analisar o potencial antiproliferativo e genotóxico de aromatizantes alimentares sintéticos, idêntico ao natural de Maracujá, e artificial de Baunilha. Esta avaliação foi realizada por meio das células meristemáticas de raízes de Allium cepa L., nos tempos de exposição de 24 e 48 horas e nas doses de 0,2; 0,4 e 0,6 ml. As raízes foram fixadas em solução de Carnoy, hidrolisadas em ácido clorídrico e coradas com orceína acética. Analisou-se, em microscópio óptico em aumento de 400×, 5.000 células por grupo tratamento, e utilizou-se o teste estatístico Qui-quadrado a 5% para análise dos dados. Verificou-se que as doses de 0,2 ml, no TE 48 h; 0,4 e 0,6 ml, nos TE 24 e 48 h, do aromatizante de Maracujá, e as três doses analisadas, nos TE 24 e 48 h, do aditivo de Baunilha reduziram significativamente o índice de divisão celular dos meristemas de raízes, mostrando-se citotóxicas. As doses 0,2; 0,4 e 0,6 ml do aditivo de Maracujá, e a de 0,6 ml do aromatizante de Baunilha, nos dois tempos de exposição considerados, induziram alterações de fuso mitótico e micronúcleos as células do organismo de prova utilizado, mostrando-se genotóxicas. Portanto, nas condições analisadas, as soluções aromatizantes de Baunilha e Maracujá, comercializadas nacional e internacionalmente, alteraram significativamente o funcionamento do ciclo celular das células meristemáticas de raízes de A. cepa.(AU)

Ellagic acid inhibits proliferation and induces apoptosis in human glioblastoma cells

PURPOSE:To investigate the anticancer activity of ellagic acid (EA) in U251 human glioblastoma cells and its possible molecular mechanism.METHODS:The cells were treated with EA at various concentrations for different time periods. Cell viability and cell proliferation were detected by cell counting kit-8(CCK-8) assay and live/dead assay respectively. Cell apoptosis were measured with Annexin V-FITC/PI double staining method by flow cytometry and Mitochondrial membrane potential assay separately. Cell cycle was measured with PI staining method by flow cytometry. The expressions of Bcl-2, Survivin, XIAP, Caspase-3, Bax, JNK, p-JNK, ERK1/2, p-ERK1/2, p38, p-p38, DR4, DR5, CHOP and GRP78-related proteins were detected by western blot after EA treatment.RESULTS:Cell viability and proliferation of glioblastoma cells treated with EA were significantly lower than the control group. EA caused robust apoptosis of the glioblastoma cells compared to the control group. EA significantly decreased the proportion at G0/G1 phases of cell cycling accompanied by increased populations at S phase in U251 cell lines. And the expressions of anti-apoptotic proteins were dramatically down-regulated.CONCLUSION:Ellagic acid potentially up-regulated DR4, DR5 and MAP kinases (JNK, ERK1/2 and p38). EA also caused significant increase in the expressions of CHOP and GRP78. Our findings suggest that EA would be beneficial for the treatment of glioblastoma.(AU)

Cytotoxicity of Cheese and Cheddar Cheese food flavorings on Allim cepa L root meristems / Citotoxicidade de aromatizantes alimentares de Queijo e Queijo Cheddar sob meristemas de raízes de Allim cepa L

Abstract Despite their great importance for the food industry, flavorings, in general, raise a number of questions regarding their cytotoxicity, mutagenicity and carcinogenicity, since, in the literature, there are few studies found evaluating the toxicity on the systemic and cellular level, of these chemical compounds. The root meristems of Allium cepa (onion) are widely used for the assessment of toxicity of chemical compounds of interest. Thus, this study aimed to evaluate, in A. cepa meristematic cells, individually and in combination at the cellular level, the toxicity of synthetic Cheese and Cheddar Cheese food flavorings, identical to the natural, at doses of 1.0 and 2.0 mL, at exposure times of 24 and 48 hours. In combination we used 0.5 mL of Cheese flavor associated with 0.5 mL of Cheddar flavor; and 1.0 mL of Cheese flavor associated with 1.0 mL of Cheddar flavor, at exposure times of 24 and 48 hours. For these evaluations, we used groups of five onion bulbs, which were first embedded in distilled water and then transferred to their respective doses. The root tips were collected and fixed in acetic acid (3:1) for 24 hours. The slides were prepared by crushing and were stained with 2% acetic orcein. Cells were analyzed throughout the cell cycle, totaling 5,000 for each control and exposure time. The mitotic indices calculated and cellular aberrations observed were subjected to statistical analysis using the chi-square test (p 0.05). No chromosomal abnormalities nor those of mitotic spindle were observed for the treatments performed. The results, both individually and in combination, showed that the flavorings under study significantly reduced the cell division rate of the test system cells used. Therefore, under the conditions studied, the two flavorings were cytotoxic.(AU)

Resumo Apesar da grande importância para a indústria alimentícia, os aromatizantes, em geral, suscitam uma série de dúvidas quanto a sua citotoxicidade, mutagenicidade e carcinogenicidade, visto que, na literatura, poucos são os trabalhos encontrados avaliando a toxicidade, em nível sistêmico e celular, destes compostos químicos. Os meristemas de raízes de Allium cepa (cebola) são muito utilizados para a avaliação da toxicidade de compostos químicos de interesse. Desta forma, este trabalho teve por objetivo avaliar em células meristemáticas de A. cepa, de forma individual, a toxicidade em nível celular de aromatizantes alimentares sintéticos, idênticos aos naturais, de sabores Queijo e Queijo Cheddar, nas doses de 1,0 e 2,0 mL, nos tempos de exposição de 24 e 48 horas; e de forma associada, onde se utilizou 0,5 mL do aromatizante sabor Queijo associado a 0,5 mL do aromatizante sabor Queijo Cheddar; e 1,0 mL do aromatizante sabor Queijo associado a 1,0 mL do aromatizante sabor Queijo Cheddar, nos tempos de exposição de 24 e 48 horas. Para estas avaliações utilizou-se grupos de cinco bulbos de cebolas, que primeiramente foram enraizados em água destilada, e em seguida transferidos para as suas respectivas doses. As radículas foram coletadas e fixadas em ácido acético (3:1) por 24 horas. As lâminas foram preparadas pela técnica de esmagamento e coradas com orceína acética a 2%. Analisaram-se células em todo ciclo celular, totalizando 5.000 para cada controle e tempo de exposição. Os índices mitóticos calculados e as aberrações celulares observadas foram submetidos à análise estatística do Qui-quadrado (p 0,05). Não foram observadas alterações cromossômicas e anomalias de fuso mitótico para nenhum dos tratamentos realizados. Os resultados obtidos, tanto individualmente como de forma associada, mostraram que os aromatizantes em estudos reduziram de forma significativa os índices de divisões celulares das células do sistema teste utilizado. Portanto, nas condições analisadas, os dois aromatizantes foram citotóxicos.(AU)

Cytotoxicity of Cheese and Cheddar Cheese food flavorings on Allim cepa L root meristems

Abstract Despite their great importance for the food industry, flavorings, in general, raise a number of questions regarding their cytotoxicity, mutagenicity and carcinogenicity, since, in the literature, there are few studies found evaluating the toxicity on the systemic and cellular level, of these chemical compounds. The root meristems of Allium cepa (onion) are widely used for the assessment of toxicity of chemical compounds of interest. Thus, this study aimed to evaluate, in A. cepa meristematic cells, individually and in combination at the cellular level, the toxicity of synthetic Cheese and Cheddar Cheese food flavorings, identical to the natural, at doses of 1.0 and 2.0 mL, at exposure times of 24 and 48 hours. In combination we used 0.5 mL of Cheese flavor associated with 0.5 mL of Cheddar flavor; and 1.0 mL of Cheese flavor associated with 1.0 mL of Cheddar flavor, at exposure times of 24 and 48 hours. For these evaluations, we used groups of five onion bulbs, which were first embedded in distilled water and then transferred to their respective doses. The root tips were collected and fixed in acetic acid (3:1) for 24 hours. The slides were prepared by crushing and were stained with 2% acetic orcein. Cells were analyzed throughout the cell cycle, totaling 5,000 for each control and exposure time. The mitotic indices calculated and cellular aberrations observed were subjected to statistical analysis using the chi-square test (p 0.05). No chromosomal abnormalities nor those of mitotic spindle were observed for the treatments performed. The results, both individually and in combination, showed that the flavorings under study significantly reduced the cell division rate of the test system cells used. Therefore, under the conditions studied, the two flavorings were cytotoxic.

Resumo Apesar da grande importância para a indústria alimentícia, os aromatizantes, em geral, suscitam uma série de dúvidas quanto a sua citotoxicidade, mutagenicidade e carcinogenicidade, visto que, na literatura, poucos são os trabalhos encontrados avaliando a toxicidade, em nível sistêmico e celular, destes compostos químicos. Os meristemas de raízes de Allium cepa (cebola) são muito utilizados para a avaliação da toxicidade de compostos químicos de interesse. Desta forma, este trabalho teve por objetivo avaliar em células meristemáticas de A. cepa, de forma individual, a toxicidade em nível celular de aromatizantes alimentares sintéticos, idênticos aos naturais, de sabores Queijo e Queijo Cheddar, nas doses de 1,0 e 2,0 mL, nos tempos de exposição de 24 e 48 horas; e de forma associada, onde se utilizou 0,5 mL do aromatizante sabor Queijo associado a 0,5 mL do aromatizante sabor Queijo Cheddar; e 1,0 mL do aromatizante sabor Queijo associado a 1,0 mL do aromatizante sabor Queijo Cheddar, nos tempos de exposição de 24 e 48 horas. Para estas avaliações utilizou-se grupos de cinco bulbos de cebolas, que primeiramente foram enraizados em água destilada, e em seguida transferidos para as suas respectivas doses. As radículas foram coletadas e fixadas em ácido acético (3:1) por 24 horas. As lâminas foram preparadas pela técnica de esmagamento e coradas com orceína acética a 2%. Analisaram-se células em todo ciclo celular, totalizando 5.000 para cada controle e tempo de exposição. Os índices mitóticos calculados e as aberrações celulares observadas foram submetidos à análise estatística do Qui-quadrado (p 0,05). Não foram observadas alterações cromossômicas e anomalias de fuso mitótico para nenhum dos tratamentos realizados. Os resultados obtidos, tanto individualmente como de forma associada, mostraram que os aromatizantes em estudos reduziram de forma significativa os índices de divisões celulares das células do sistema teste utilizado. Portanto, nas condições analisadas, os dois aromatizantes foram citotóxicos.

Antitumor potential of the myotoxin BthTX-I from Bothrops jararacussu snake venom: evaluation of cell cycle alterations and death mechanisms induced in tumor cell lines

Background Phospholipases A 2 (PLA 2 s) are abundant components of snake venoms that have been extensively studied due to their pharmacological and pathophysiological effects on living organisms. This study aimed to assess the antitumor potential of BthTX-I, a basic myotoxic PLA 2isolated from Bothrops jararacussu venom, by evaluating in vitro processes of cytotoxicity, modulation of the cell cycle and induction of apoptosis in human (HL-60 and HepG2) and murine (PC-12 and B16F10) tumor cell lines. Methods The cytotoxic effects of BthTX-I were evaluated on the tumor cell lines HL-60 (promyelocytic leukemia), HepG2 (human hepatocellular carcinoma), PC-12 (murine pheochromocytoma) and B16F10 (murine melanoma) using the MTT method. Flow cytometry technique was used for the analysis of cell cycle alterations and death mechanisms (apoptosis and/or necrosis) induced in tumor cells after treatment with BthTX-I. Results It was observed that BthTX-I was cytotoxic to all evaluated tumor cell lines, reducing their viability in 40 to 50 %. The myotoxin showed modulating effects on the cell cycle of PC-12 and B16F10 cells, promoting delay in the G0/G1 phase. Additionally, flow cytometry analysis indicated cell death mainly by apoptosis. B16F10 was more susceptible to the effects of BthTX-I, with ~40 % of the cells analyzed in apoptosis, followed by HepG2 (~35 %), PC-12 (~25 %) and HL-60 (~4 %). Conclusions These results suggest that BthTX-I presents antitumor properties that may be useful for developing new therapeutic strategies against cancer.(AU)

Antitumor potential of the myotoxin BthTX-I from Bothrops jararacussu snake venom: evaluation of cell cycle alterations and death mechanisms induced in tumor cell lines

Background Phospholipases A 2 (PLA 2 s) are abundant components of snake venoms that have been extensively studied due to their pharmacological and pathophysiological effects on living organisms. This study aimed to assess the antitumor potential of BthTX-I, a basic myotoxic PLA 2isolated from Bothrops jararacussu venom, by evaluating in vitro processes of cytotoxicity, modulation of the cell cycle and induction of apoptosis in human (HL-60 and HepG2) and murine (PC-12 and B16F10) tumor cell lines. Methods The cytotoxic effects of BthTX-I were evaluated on the tumor cell lines HL-60 (promyelocytic leukemia), HepG2 (human hepatocellular carcinoma), PC-12 (murine pheochromocytoma) and B16F10 (murine melanoma) using the MTT method. Flow cytometry technique was used for the analysis of cell cycle alterations and death mechanisms (apoptosis and/or necrosis) induced in tumor cells after treatment with BthTX-I. Results It was observed that BthTX-I was cytotoxic to all evaluated tumor cell lines, reducing their viability in 40 to 50 %. The myotoxin showed modulating effects on the cell cycle of PC-12 and B16F10 cells, promoting delay in the G0/G1 phase. Additionally, flow cytometry analysis indicated cell death mainly by apoptosis. B16F10 was more susceptible to the effects of BthTX-I, with ~40 % of the cells analyzed in apoptosis, followed by HepG2 (~35 %), PC-12 (~25 %) and HL-60 (~4 %). Conclusions These results suggest that BthTX-I presents antitumor properties that may be useful for developing new therapeutic strategies against cancer.(AU)

Antitumor potential of the myotoxin BthTX-I from Bothrops jararacussu snake venom: evaluation of cell cycle alterations and death mechanisms induced in tumor cell lines

Background Phospholipases A 2 (PLA 2 s) are abundant components of snake venoms that have been extensively studied due to their pharmacological and pathophysiological effects on living organisms. This study aimed to assess the antitumor potential of BthTX-I, a basic myotoxic PLA 2isolated from Bothrops jararacussu venom, by evaluating in vitro processes of cytotoxicity, modulation of the cell cycle and induction of apoptosis in human (HL-60 and HepG2) and murine (PC-12 and B16F10) tumor cell lines. Methods The cytotoxic effects of BthTX-I were evaluated on the tumor cell lines HL-60 (promyelocytic leukemia), HepG2 (human hepatocellular carcinoma), PC-12 (murine pheochromocytoma) and B16F10 (murine melanoma) using the MTT method. Flow cytometry technique was used for the analysis of cell cycle alterations and death mechanisms (apoptosis and/or necrosis) induced in tumor cells after treatment with BthTX-I. Results It was observed that BthTX-I was cytotoxic to all evaluated tumor cell lines, reducing their viability in 40 to 50 %. The myotoxin showed modulating effects on the cell cycle of PC-12 and B16F10 cells, promoting delay in the G0/G1 phase. Additionally, flow cytometry analysis indicated cell death mainly by apoptosis. B16F10 was more susceptible to the effects of BthTX-I, with ~40 % of the cells analyzed in apoptosis, followed by HepG2 (~35 %), PC-12 (~25 %) and HL-60 (~4 %). Conclusions These results suggest that BthTX-I presents antitumor properties that may be useful for developing new therapeutic strategies against cancer.

Efeitos da morfina e metadona sobre o comportamento e crescimento tumoral de camundongos com tumor de Ehrlich

RUSCH, E. Efeitos da morfina e metadona sobre o comportamento e crescimento tumoral de camundongos com tumor de Ehrlich. 2020. Dissertação (Mestrado). Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, Pirassununga, 2020. A morfina e a metadona, embora sejam fármacos recomendados para promover analgesia, parecem promover alterações comportamentais e influenciar no crescimento tumoral em modelos experimentais. Diante disso, o objetivo do estudo consistiu em avaliar os efeitos da morfina e metadona sobre o comportamento, antinocicepção e crescimento tumoral em camundongos. O estudo foi dividido em duas partes. No primeiro experimento foram utilizados 53 camundongos, fêmeas, com 60 ± 10 dias de idade que foram inoculados com tumor ascítico de Ehrlich (TAE) por via intraperitoneal. Após sete dias da inoculação, os animais foram distribuídos aleatoriamente em 7 grupos, morfina 5 mg/kg (Morf5), morfina 7,5 mg/kg (Morf7,5), morfina 10 mg/kg (Morf10), metadona 2,85 mg/kg (Met2,85), metadona 4,3 mg/kg (Met4,3), metadona 5,7 mg/kg (Met5,7) e solução salina NaCl 0,9% (Salina). Os tratamentos foram administrados por via subcutânea, a cada seis horas, durante três dias. Os animais foram avaliados quanto a atividade geral em campo aberto e nocicepção, por meio do teste de pinçamento de cauda, os quais foram realizados antes da inoculação tumoral (dia 0), aos 40, 90, 150, 240 e 360 minutos após o início dos tratamentos (dia 7) e 40, 150 e 360 minutos após os dias 8 e 9 pós-inoculação. Todas as doses promoveram aumento significativo da distância percorrida e velocidade média de maneira dose-dependente, sendo que os efeitos foram mais pronunciados nos dias 8 e 9. As frequências de levantar e de autolimpeza reduziram de maneira significativa após a administração de morfina e metadona em todas as doses até os 90 minutos. O segundo experimento consistiu em avaliar os efeitos sobre o crescimento tumoral das mesmas doses da morfina e metadona administradas por via subcutânea, a cada 6 horas, durante 8 dias, iniciados 24 horas após a inoculação tumoral. Os animais foram avaliados diariamente quanto ao peso e circunferência abdominal e nove dias após a inoculação tumoral, foram submetidos à eutanásia. O líquido ascítico foi colhido para aferição do volume, verificação da característica do líquido, contagem de células tumorais e análise do ciclo celular. Todos os animais apresentaram aumento de peso e de circunferência abdominal ao longo dos dias. O volume do líquido ascítico peritoneal foi menor nos tratamentos Morf5, Morf10 e em todas as doses testadas de metadona em comparação ao grupo Salina. A viabilidade e o número de células totais não diferiram entre os tratamentos. Os tratamentos Morf10 e Met5,7 interferiram no ciclo celular das células tumorais com maior porcentagem de células na fase G1 do ciclo, em comparação ao grupo Salina. Observou-se, a partir do primeiro experimento que todas as doses testadas promovem aumento da locomoção e redução de comportamentos exploratórios que foram mais evidentes ao longo dos dias de tratamento. A antinocicepção é observada por até 40 minutos em dose única e prolonga-se por até 150 minutos após administrações seriadas nas doses intermediárias e maiores. Os tratamentos Morf10 e Met5,7 promovem estase do ciclo celular, mas não interferem de maneira significativa no crescimento do tumor ascítico de Ehrlich.

RUSCH, E. Effects of morphine and methadone on the behavior and tumor growth of mice with Ehrlich tumor. 2020. 26 f. M.Sc. Dissertation - Faculdade de Zootecnia e Engenharia de Alimentos, University of Sao Paulo, Pirassununga, 2020. The objective of this research is to evaluate the effects of morphine and methadone on behavior, antinociception and tumor growth in mice. The study was dived into two parts. In the first experiment, Fifty-three female mice, 60 ± 10 days old, were inoculated with Ehrlich's ascitic tumor (EAT) intraperitoneally. Seven days after intraperitoneal tumour inoculation (2 × 106 cells), the animals were randomised into seven groups: morphine 5 mg/kg (MO5), morphine 7.5 mg/kg (MO7.5), morphine 10 mg/kg (MO10), methadone 2.85 mg/kg (ME2.85), methadone 4.3 mg/kg (ME4.3), methadone 5.7 mg/kg (ME5.7), and 0.9% NaCl (Sal). Treatments were administered subcutaneously, every 6 h, for 3 days. The animals were evaluated for general activity and nociception using the open field and tail clip tests, respectively. These tests were performed before tumour inoculation (day 0); at 40, 90, 150, 240, and 360 min following treatment initiation (day 7); and at 40, 150, and 360 min after days 8 and 9 post-inoculation. All evaluated doses promoted a significant dose-dependent increase in the total distance travelled and the average speed, markedly pronounced on days 8 and 9 than on day 7. The frequencies of rearing and self-grooming decreased significantly after morphine or methadone administration. The second experiment consisted of evaluating the effects on tumor growth of the same doses of morphine and methadone administered subcutaneously, every 6 hours, for 8 days, starting 24 hours after tumor inoculation. The animals were evaluated daily for weight and abdominal circumference and they were euthanized nine days after the tumor inoculation. The ascitic fluid was collected to measure the volume, check the characteristic of the liquid, count the tumor cells and analyze the cell cycle. All animals had increased weight and waist circumference over the days. The volume of peritoneal ascitic fluid was lower in the Morf5, Morf10 treatments and in all tested methadone doses compared to the Saline group. Viability and number of total cells did not differ between treatments. Morf10 and Met5,7 treatments interfered in the cell cycle of tumor cells, with a higher percentage of cells in the G1 phase of the cycle, compared to the Saline group. It was observed, from the first experiment, that all doses tested promote increased locomotion and reduced exploratory behaviors that were more evident over the treatment days. Antinociception was observed for up to 40 minutes in a single dose and continued for up to 150 minutes after serial administrations in intermediate and higher doses. The Morf10 and Met5,7 treatments promoted cell cycle stasis, but did not significantly interfere with the growth of Ehrlich's ascites tumor.

ESTABELECIMENTO DE CARIOPLASTOS E CITOPLASTOS DE CATETOS, Pecari tajacu (LINNAEUS, 1758), VISANDO A CLONAGEM POR TRANSFERÊNCIA NUCLEAR DE CÉLULA SOMÁTICA EM TAIASSUÍDEOS

O direcionamento dos primeiros passos para a realização da transferência nuclear de célula somática (TNCS) em catetos garantirá uma ferramenta efetiva na conservação da espécie, perante sua acelerada diminuição populacional e sua atividade ecológica indispensável para o ecossistema. Para tanto, a presente tese foi dividida em duas etapas (três experimentos por etapa), sendo a primeira etapa o estudo das células doadoras de núcleo ou carioplastos e a segunda etapa, o estudo das células doadoras de citoplasma ou citoplastos. Assim, diante da importância de carioplastos de qualidade reconhecida para a TNCS, nós inicialmente estabelecemos cinco linhagens de fibroblastos de catetos, monitorando a viabilidade, atividade metabólica e estresse oxidativo, de acordo com os efeitos do número de passagens (primeira, terceira e décima) e criopreservação. Embora não haja efeito desses critérios sobre a viabilidade, células em décima passagem tiveram uma redução de seu metabolismo. Adicionalmente, células congeladas/descongeladas tiveram um aumento no número de espécies reativas de oxigênio e potencial de membrana mitocondrial. Além disso, sabendo da importância de manter estas células armazenadas em um biobanco de maneira adequada, nós otimizamos a solução crioprotetora utilizada na congelação lenta de fibroblastos de catetos. Deste modo, a solução composta por 10% de dimetilsulfóxido (DMSO) com 0,2 M de sacarose e 50% de soro fetal bovino (SFB) foi considerada a solução mais eficiente em manter a viabilidade, atividade proliferativa, metabolismo e níveis adequados de estresse oxidativo de células somáticas de catetos, quando comparada a soluções na ausência de sacarose e com 10% de SFB em diferentes combinações. Finalmente, um passo essencial no estabelecimento dos carioplastos para a TNCS consiste na sincronização das células em G0/G1 do ciclo celular. Deste modo, nós avaliamos diferentes métodos de sincronização do ciclo celular: (i) supressão de soro (SS) por um a quatro dias, (ii) inibição por contato (IC) por um a três dias e (iii) agentes químicos [DMSO, 6-dimetilaminopurina (6-DMAP), ciclohexamida (CHX), e citocalasina B (CB)] por um a dois dias, em termos de seus efeitos sobre a sincronização em G0/G1 e viabilidade. Assim, nós observamos que a IC por três dias foi o método mais eficiente para sincronização do ciclo celular e manutenção da viabilidade de fibroblastos de catetos. Portanto, com estes três experimentos, nós estabelecemos a etapa de carioplastos da TNCS de catetos, obtendo células de qualidade e aptas a serem usadas como doadoras de núcleo. Na segunda etapa, nós, inicialmente, adequamos as condições de maturação in vitro (MIV) de oócitos de catetos, avaliando o tempo de MIV e o efeito do fator de crescimento epidermal (EGF) sobre a habilidade meiótica. Assim, nós concluímos que 48 h é o período adequado para a MIV de oócitos quando comparado ao tempo de 24 h, de acordo com o potencial meiótico. Ainda, observou-se que o EGF pode ser utilizado para otimizar o meio de MIV. Finalmente, no terceiro experimento, nós avaliamos a habilidade de desenvolvimento destes oócitos após ativação artificial, usando a ionomicina como ativador primário e comparando diferentes ativadores secundários (6-DMAP, CHX e CB). Nós verificamos que a ativação química usando ionomicina e 6-DMAP foi a mais eficiente combinação, tendo esta tese alcançado como resultado significativo, uma taxa de 27,6% de blastocistos de catetos derivados da ativação oocitária artificial. Em síntese, nós obtivemos carioplastos e citoplastos que poderão ser empregados na TNCS de catetos, deixando a ponto as etapas fundamentais para a clonagem desta espécie. Ainda, destaca-se que os conhecimentos aqui gerados poderão ser aplicados em estudos de fecundação in vitro, compreensão do desenvolvimento embrionário, produção de células induzidas à pluripotência, e ensaios de toxicidade. Portanto, este trabalho foi um grande passo para a conservação de catetos.

The direction of the first steps for the achievement of somatic cell nuclear transfer (SCNT) in collared peccary will guarantee an effective tool in the conservation of the species, in view of its accelerated population decrease and its essential ecological activity for the ecosystem. Therefore, the present thesis was divided into two steps (three experiments per step), being the first step the study of the donor cells of nucleus or karyoplast and the second stage, the study of the donor cells of cytoplasm or cytoplasts. Thus, in view of the importance of acknowledge quality karyoplast for SCNT, we initially established five cell lines of collared peccary fibroblasts, monitoring viability, metabolic activity and oxidative stress, according to the effects of the number of passages (first, third and tenth) and cryopreservation. Although there is no effect of these criteria on viability, cells in tenth passage had a reduction in their metabolism. Additionally, frozen/thawed cells had an increase in the number of reactive oxygen species and mitochondrial membrane potential. Moreover, knowing the importance of maintaining these cells stored in a biobank properly, we optimize the cryoprotectant solution used in the slow freezing of collared peccary fibroblasts. Thus, the solution composed of 10% dimethyl sulfoxide (DMSO) with 0.2 M sucrose and 50% fetal bovine serum (FBS) was considered the most efficient solution in maintaining the viability, proliferative activity, metabolism and adequate levels of oxidative stress of somatic cell cells, when compared to solutions in the absence of sucrose and with 10% FBS in different combinations. Finally, an essential step in establishing the karyoplast for SCNT is the synchronization of cells in G0/G1 of the cell cycle. Thus, we evaluated different cell cycle synchronization methods: (i) serum suppression (SS) for one to four days, (ii) contact inhibition (CI) for one to three days and (iii) chemical agents [DMSO, 6-dimethylaminopurine (6-DMAP), cyclohexamide (CHX), and cytochalasin B (CB)] for one to two days, in terms of their effects on G0/G1 synchronization and viability. Thus, we observed that the IC for three days was the most efficient method for synchronizing the cell cycle and maintaining the viability of collared peccary fibroblasts. Consequently, with these three experiments, we have established karyoplast stage of SCNT in collared peccary, obtaining quality cells and able to be used as nuclear donors. In the second stage, we initially adjusted the in vitro maturation (IVM) conditions of collared peccary oocytes, evaluating the IVM time and the effect of the epidermal growth factor (EGF) on the meiotic ability. Thus, we concluded that 48 h is the appropriate period for oocyte IVM when compared to 24 h, according to meiotic potential. Still, it was observed that EGF can be used to optimize the IVM medium. Finally, in the third experiment, we evaluated the developmental ability of these oocytes after artificial activation, using ionomycin as the primary activator and comparing different secondary activators (6-DMAP, CHX and CB). We found that chemical activation using ionomycin and 6-DMAP was the most efficient combination, with this thesis achieving as a significant result, a rate of 27.6% of blastocysts of collared peccaries derived from oocyte artificial activation. In summary, we got karyoplast and cytoplasts that may be employed in the SCNT of collared peccary, leaving the point the fundamental steps for the cloning of this species. Furthermore, it is emphasized that the knowledge generated here can be applied for in vitro fertilization, studies understanding of embryonic development, production cells induced to pluripotency, and toxicity assessments. Therefore, this work was a great step for the conservation of collared peccaries.

Isolamento e caracterização de células doadoras derivadas da pele para a transferência nuclear / Isolation and characterization of skin-derived donor cells for nuclear transfer

O sucesso das biotecnologias reprodutivas assistidas, especialmente a clonagem por transferência nuclear de células somáticas, envolve múltiplos fatores e cada um destes pode influenciar a eficiência final. Em geral, os protocolos de isolamento, caracterização e criopreservação de células das espécies a serem clonadas constituem a etapa inicial para a obtenção de descendência clone viável, além de contribuir para outros estudos relacionados à biotecnologia celular. Neste contexto, o sucesso desta etapa pode ser alcançado pela associação da escolha do tipo celular, estabelecimento de altas taxas de viabilidade, atividade proliferativa e metabólica após o cultivo in vitro e criopreservação, além da sincronia do ciclo celular antes da reconstrução do embrião clone. Os fibroblastos e outras células derivadas da pele oriundas da região auricular de fetos ou animais adultos representam a fonte de fácil manipulação e mais adequada para a obtenção de carioplastos. Adicionalmente, avanços nesta área têm mostrado a possibilidade da produção de células pluripotentes a partir de células somáticas modificadas. Assim, o objetivo deste manuscrito é realizar uma revisão sobre os aspectos práticos da obtenção e estabelecimento de células doadoras de núcleo derivadas da pele para a reconstrução embrionária, descrevendo os fatores relacionados em cada procedimento que interferem no resultado final.(AU)

The success of assisted reproductive biotechnology, especially cloning by somatic cell nuclear transfer, involves multiple factors and each of these may influence the final efficiency. In general, the protocols for the isolation, characterization and cryopreservation of cells of the species to be cloned consist in initial step for obtaining viable clone offspring, and contribute to other studies on cell biotechnology. In this context, the success of this step may be achieved by association of the choice of cell type, establishment of high rates of viability, metabolic and proliferative activities after in vitro culture and cryopreservation, and cell cycle synchronization prior to embryo reconstruction. Fibroblasts and other skin derived-cells from ear region of fetal or adult animals represent a source of easy handling and better appropriate for obtaining caryoplasts. Additionally, advances in this area are showing the possibility of induced pluripotent cells from modified somatic cells. Thus, the aim of this manuscript is to describe the practical aspects of obtaining and establishing skin-derived donor cells for embryo reconstruction, with emphasis to the factors listed in each procedure that affect the final outcome.(AU)