O desenvolvimento de novas estratégias terapêuticas ao melanoma é de particular importância devido à sua baixa resposta aos tratamentos tradicionais. No presente trabalho, utilizamos modelo de melanoma murino experimental para estudarmos os efeitos da fosfoetanolamina (PEA) sintética sobre o desenvolvimento deste tumor. Nossos resultados demonstram que o fosfomonoéster apresentou efeito inibidor da proliferação de células da linhagem B16F10 in vitro, induzindo apoptose após estimulação por 24 a 72h. In vivo, o tratamento (via oral) de animais portadores de melanoma com diferentes doses de PEA (10, 20 e 40mg/Kg), durante 10 ou 20 dias consecutivos, resultou em volumes tumorais pelo menos 70% menores que o de animais controle e diferenças macroscópicas consideráveis. PEA induziu, de maneira dose-dependente, aumento da apoptose e diminuição da proliferação de células tumorais. O tratamento resultou em alterações hematológicas como aumento do número de plaquetas, eritrócitos e leucócitos. Dentre os leucócitos, observou-se uma maior proporção de linfócitos e monócitos após 10 e 20 dias de tratamento, respectivamente. Em adição, PEA induziu uma maior produção da citocina pró-inflamatória IL-6 e das citocinas anti-inflamatórias IL-10 e TGF- e menores níveis da citocina pró-inflamatória IFN-. Os níveis de IL-1, IL-12p70 e IL-17 não foram alterados com o tratamento. Nossos resultados demonstram um papel inibidor da PEA sobre a progressão do melanoma, contribuindo para um melhor entendimento de sua atividade anti-tumoral. / The low responsiveness of melanoma to traditional treatments together with its increasing incidence makes the development of new therapeutic strategies against this type of cancer extremely important. In this study, we used a murine melanoma model to evaluate the effects of synthetic phosphoethanolamine (PEA) on the development of this tumor. In vitro, PEA had an inhibitory effect on the proliferation of B16F10 cells, inducing apoptosis after 24 to 72h stimulation. In vivo, oral treatment of melanoma-bearing animals with different doses of PEA (10, 20 e 40mg/Kg) during 10 or 20 consecutive days resulted in reduced tumor volumes (at least 70% compared to the control) and in expressive macroscopic differences. PEA also induced a dose-dependent increase of apoptosis and decrease in tumor cell proliferation. The treatment also resulted in hematological changes, such as increased numbers of platelets, erythrocytes and leukocytes. Among leukocytes, we observed a higher proportion of lymphocytes and monocytes after 10 and 20 days of treatment, respectively. In addition, PEA induced higher levels of the pro-inflammatory cytokine IL-6 and of the anti-inflammatory cytokines IL-10 and TGF-, and it also induced a lower production of the pro-inflammatory cytokine IFN-. No differences were observed in the levels of IL-1, TNF-, IL-12p70 and IL-17 upon treatment. Our results demonstrate an inhibitory role of PEA in the development of melanoma, contributing to a better understanding of its antitumoral activity.

O desenvolvimento de novas estratégias terapêuticas ao melanoma é de particular importância devido à sua baixa resposta aos tratamentos tradicionais. No presente trabalho, utilizamos modelo de melanoma murino experimental para estudarmos os efeitos da fosfoetanolamina (PEA) sintética sobre o desenvolvimento deste tumor. Nossos resultados demonstram que o fosfomonoéster apresentou efeito inibidor da proliferação de células da linhagem B16F10 in vitro, induzindo apoptose após estimulação por 24 a 72h. In vivo, o tratamento (via oral) de animais portadores de melanoma com diferentes doses de PEA (10, 20 e 40mg/Kg), durante 10 ou 20 dias consecutivos, resultou em volumes tumorais pelo menos 70% menores que o de animais controle e diferenças macroscópicas consideráveis. PEA induziu, de maneira dose-dependente, aumento da apoptose e diminuição da proliferação de células tumorais. O tratamento resultou em alterações hematológicas como aumento do número de plaquetas, eritrócitos e leucócitos. Dentre os leucócitos, observou-se uma maior proporção de linfócitos e monócitos após 10 e 20 dias de tratamento, respectivamente. Em adição, PEA induziu uma maior produção da citocina pró-inflamatória IL-6 e das citocinas anti-inflamatórias IL-10 e TGF- e menores níveis da citocina pró-inflamatória IFN-. Os níveis de IL-1, IL-12p70 e IL-17 não foram alterados com o tratamento. Nossos resultados demonstram um papel inibidor da PEA sobre a progressão do melanoma, contribuindo para um melhor entendimento de sua atividade anti-tumoral. / The low responsiveness of melanoma to traditional treatments together with its increasing incidence makes the development of new therapeutic strategies against this type of cancer extremely important. In this study, we used a murine melanoma model to evaluate the effects of synthetic phosphoethanolamine (PEA) on the development of this tumor. In vitro, PEA had an inhibitory effect on the proliferation of B16F10 cells, inducing apoptosis after 24 to 72h stimulation. In vivo, oral treatment of melanoma-bearing animals with different doses of PEA (10, 20 e 40mg/Kg) during 10 or 20 consecutive days resulted in reduced tumor volumes (at least 70% compared to the control) and in expressive macroscopic differences. PEA also induced a dose-dependent increase of apoptosis and decrease in tumor cell proliferation. The treatment also resulted in hematological changes, such as increased numbers of platelets, erythrocytes and leukocytes. Among leukocytes, we observed a higher proportion of lymphocytes and monocytes after 10 and 20 days of treatment, respectively. In addition, PEA induced higher levels of the pro-inflammatory cytokine IL-6 and of the anti-inflammatory cytokines IL-10 and TGF-, and it also induced a lower production of the pro-inflammatory cytokine IFN-. No differences were observed in the levels of IL-1, TNF-, IL-12p70 and IL-17 upon treatment. Our results demonstrate an inhibitory role of PEA in the development of melanoma, contributing to a better understanding of its antitumoral activity.

Indiana University-Purdue University Indianapolis (IUPUI) / Haemophilus ducreyi resists the cytotoxic effects of human antimicrobial peptides (APs), including α-defensins, β-defensins, and the cathelicidin LL-37. Resistance to LL-37, mediated by the sensitive to antimicrobial peptide (Sap) transporter, is required for H. ducreyi virulence in humans. Cationic APs are attracted to the negatively charged bacterial cell surface. In other gram-negative bacteria, modification of lipopolysaccharide or lipooligosaccharide (LOS) by the addition of positively charged moieties, such as phosphoethanolamine (PEA), confers AP resistance by means of electrostatic repulsion. H. ducreyi LOS has PEA modifications at two sites, and we identified three genes (lptA, ptdA, and ptdB) in H. ducreyi with homology to a family of bacterial PEA transferases. We generated non-polar, unmarked mutants with deletions in one, two, or all three putative PEA transferase genes. Mutants with deletions in two PEA transferase genes were significantly more susceptible to β-defensins, and the triple mutant was significantly more susceptible to both α- and β-defensins, but not LL-37; complementation of all three genes restored parental levels of AP resistance. Deletion of all three PEA transferase genes also resulted in a significant increase in the negativity of the mutant cell surface, suggesting these three genes contribute to the addition of positively charged moieties on the cell surface. Mass spectrometric analysis revealed that LptA was required for PEA modification of lipid A; PtdtA and PtdB did not affect PEA modification of LOS. In human inoculation experiments, the triple mutant was as virulent as its parent strain. While this is the first identified mechanism of resistance to α-defensins in H. ducreyi, our in vivo data suggest that resistance to cathelicidin may be more important than defensin resistance to H. ducreyi pathogenesis.

No presente trabalho foram realizados estudos de equilíbrios químicos da fosfoetanolamina (FOS) ou 2-aminoetanol-dihidrogenofosfato, substância produzida no organismo humano, um precursor de fosfatidiletanolamina importante por regularizar o metabolismo de disfunção celular e metabólica. A constante de ionização de uma molécula é uma ferramenta interessante a fim de prever a sua absorção no organismo que geralmente, apenas ocorre com as moléculas na forma não ionizada. Na determinação usual de constantes de zwiterions como a FOS, não se considera o equilíbrio de formação de espécies não ionizadas como dímero (agregado). Neste trabalho comprovou-se o fenômeno de agregação da FOS por titulações potenciométricas e pelo método espectrofotométrico Azul de Molibdênio. Determinou-se a constante de agregação pelo método potenciométrico como sendo da ordem de 106. Realizou-se a determinação da primeira constante de ionização da FOS por titulações em meio ácido, na ausência do fenômeno de agregação, e verificou-se que é da ordem de 10-4. Pelo método espectrofotométrico Azul de Molibdênio determinou-se a porcentagem de fosfato iônico 1%, coerente com os resultados potenciométricos. A determinação da segunda constante de ionização da FOS foi realizada por titulações com monoetanolamina (MEA), uma base fraca. Verificou-se a possibilidade de utilização do sistema FOS/MEA como um tampão biológico, por meio do gráfico de máxima capacidade tamponante vs pH. A determinação de terceira constante de ionização da FOS foi realizada por meio de titulações com NaOH na ausência e na presença de formaldeído, bloqueando o grupo amino. A determinação simultânea das três constantes de ionização foi realizada por meio de uma titulação de retorno,com HCl e adição de NaOH. Com o tratamento matemático desta curva, determinou-se o número médio de protonação e verificaram-se as regiões de máxima capacidade tamponante. Obteve-se a curva de distribuição de espécies e o diagrama logarítmico para a FOS com os valores de constante de ionização determinados na ausência do fenômeno de agregação. Estudou-se a interação de íons cálcio e magnésio com a FOS, verificou-se que os complexos são do tipo ML e M2L. É importante salientar ainda, que não há relato na literatura desses estudos . / Studies on the chemical equilibrium of phosphoethanolamine (PEA) or 2-aminoethanol-dihydrogen phosphate, produced by the human body, as an important phosphatydilethanolamine precursor that regularizes the cell and metabolic disfuntion have been performed. The ionization constant is an interesting tool to foresee organic absortion which often occurs in non ionized form. In the usual determination of zwiterion, constants like PEA, the dimmer formation equilibrium is not always considered. In this work aggregation phenomena of PEA has been demonstrated by potentiometric titration and molybdenium blue spectrophotometric method. The aggregation constant was determinated by potentiometric method as 106. The first ionization constant determination of PEA was made by titration in acidic medium without aggregation phenomena and it was 10-4. The molybdenum blue spectrophotometric method was used to determine the free phosphate amount, 1%, was coherent with potentiometric results. The second ionization constant determination was made by titration with monoethanolamine (MEA), a weak base. There is the possibility of using the system PEA/MEA as a biological buffer, according to the maximum buffer capacity graphic versus pH. The third ionization constant determination was made by titration with NaOH, with and without formaldehyde, blocking the amino group. The simultaneous determination of the three constants of ionization was made by back titration, with HCl after the addition of NaOH. The average number of bound protons was done with mathematical treatment of back titration curve and then, the maximum buffer capacity regions were observed. The species distribution curve and logarithmic diagram were obtained using the ionization constant values determined in the aggregation phenomena absence. The interaction between calcium, magnesium and PEA was studied and the ML and M2L species were observed, not described in literature yet.

No presente trabalho foram realizados estudos de equilíbrios químicos da fosfoetanolamina (FOS) ou 2-aminoetanol-dihidrogenofosfato, substância produzida no organismo humano, um precursor de fosfatidiletanolamina importante por regularizar o metabolismo de disfunção celular e metabólica. A constante de ionização de uma molécula é uma ferramenta interessante a fim de prever a sua absorção no organismo que geralmente, apenas ocorre com as moléculas na forma não ionizada. Na determinação usual de constantes de zwiterions como a FOS, não se considera o equilíbrio de formação de espécies não ionizadas como dímero (agregado). Neste trabalho comprovou-se o fenômeno de agregação da FOS por titulações potenciométricas e pelo método espectrofotométrico Azul de Molibdênio. Determinou-se a constante de agregação pelo método potenciométrico como sendo da ordem de 106. Realizou-se a determinação da primeira constante de ionização da FOS por titulações em meio ácido, na ausência do fenômeno de agregação, e verificou-se que é da ordem de 10-4. Pelo método espectrofotométrico Azul de Molibdênio determinou-se a porcentagem de fosfato iônico 1%, coerente com os resultados potenciométricos. A determinação da segunda constante de ionização da FOS foi realizada por titulações com monoetanolamina (MEA), uma base fraca. Verificou-se a possibilidade de utilização do sistema FOS/MEA como um tampão biológico, por meio do gráfico de máxima capacidade tamponante vs pH. A determinação de terceira constante de ionização da FOS foi realizada por meio de titulações com NaOH na ausência e na presença de formaldeído, bloqueando o grupo amino. A determinação simultânea das três constantes de ionização foi realizada por meio de uma titulação de retorno,com HCl e adição de NaOH. Com o tratamento matemático desta curva, determinou-se o número médio de protonação e verificaram-se as regiões de máxima capacidade tamponante. Obteve-se a curva de distribuição de espécies e o diagrama logarítmico para a FOS com os valores de constante de ionização determinados na ausência do fenômeno de agregação. Estudou-se a interação de íons cálcio e magnésio com a FOS, verificou-se que os complexos são do tipo ML e M2L. É importante salientar ainda, que não há relato na literatura desses estudos . / Studies on the chemical equilibrium of phosphoethanolamine (PEA) or 2-aminoethanol-dihydrogen phosphate, produced by the human body, as an important phosphatydilethanolamine precursor that regularizes the cell and metabolic disfuntion have been performed. The ionization constant is an interesting tool to foresee organic absortion which often occurs in non ionized form. In the usual determination of zwiterion, constants like PEA, the dimmer formation equilibrium is not always considered. In this work aggregation phenomena of PEA has been demonstrated by potentiometric titration and molybdenium blue spectrophotometric method. The aggregation constant was determinated by potentiometric method as 106. The first ionization constant determination of PEA was made by titration in acidic medium without aggregation phenomena and it was 10-4. The molybdenum blue spectrophotometric method was used to determine the free phosphate amount, 1%, was coherent with potentiometric results. The second ionization constant determination was made by titration with monoethanolamine (MEA), a weak base. There is the possibility of using the system PEA/MEA as a biological buffer, according to the maximum buffer capacity graphic versus pH. The third ionization constant determination was made by titration with NaOH, with and without formaldehyde, blocking the amino group. The simultaneous determination of the three constants of ionization was made by back titration, with HCl after the addition of NaOH. The average number of bound protons was done with mathematical treatment of back titration curve and then, the maximum buffer capacity regions were observed. The species distribution curve and logarithmic diagram were obtained using the ionization constant values determined in the aggregation phenomena absence. The interaction between calcium, magnesium and PEA was studied and the ML and M2L species were observed, not described in literature yet.

Modification of the lipid A domain of lipopolysaccharide (LPS) has been reported to contribute to the virulence and pathogenesis of various Gram-negative bacteria. The Kdo (3-deoxy-D-manno-octulosonic acid)-lipid A domain of Helicobacter pylori LPS shows several differences to that of Escherichia coli. It has fewer acyl chains, a reduced number of phosphate groups, much lower immunobiological activity, and only a single Kdo sugar is attached to the disaccharide backbone. However, H. pylori synthesizes a minor lipid A species resembling that of E. coli, which is both bis-phosphorylated and hexa-acylated suggesting that the major species results from the action of specific modifying enzymes. This work describes two enzymes, a lipid A phosphatase and a phosphoethanolamine transferase, involved in the periplasmic modification of the 1-position of H. pylori lipid A. Furthermore, we report a novel Kdo trimming enzyme that requires prior removal of the 1-phosphate group for enzymatic activity. Discovery of the enzymatic machinery involved in the remodeling of H. pylori LPS will help unravel the importance of these modifications in H. pylori pathogenesis.d.

C-reactive protein (CRP) is an evolutionarily conserved protein, a component of the innate immune system, and an acute phase protein in humans. In addition to its raised level in blood in inflammatory states, CRP is also localized at sites of inflammation including atherosclerotic lesions, arthritic joints and amyloid plaque deposits. Results of in vivo experiments in animal models of inflammatory diseases indicate that CRP is an anti-pneumococcal, anti-atherosclerotic, anti-arthritic and an anti-amyloidogenic molecule. The mechanisms through which CRP functions in inflammatory diseases are not fully defined; however, the ligand recognition function of CRP in its native and non-native pentameric structural conformations and the complement-activating ability of ligand-complexed CRP have been suggested to play a role. One tool to understand the structure-function relationships of CRP and determine the contributions of the recognition and effector functions of CRP in host defense is to employ site-directed mutagenesis to create mutants for experimentation. For example, CRP mutants incapable of binding to phosphocholine are generated to investigate the importance of the phosphocholine-binding property of CRP in mediating host defense. Recombinant CRP mutants can be expressed in mammalian cells and, if expressed, can be purified from the cell culture media. While the methods to purify wild-type CRP are well established, different purification strategies are needed to purify various mutant forms of CRP if the mutant does not bind to either calcium or phosphocholine. In this article, we report the methods used to purify pentameric recombinant wild-type and mutant CRP expressed in and secreted by mammalian cells.

The compatible solute glycine betaine accumulates in many plants including spinach (Spinacea oleracea) under conditions of water deficit stress. The precursor to glycine betaine is choline, a ubiquitous metabolite in plants as a component of phosphotidylcholine. In spinach choline is synthesized from phosphocholine, a product of three sequential N-methylations of phosphoethanolamine catalysed by the cytosolic enzyme S-adenosyl-L-methionine: phosphoethanolamine-N-methyltransferase (PEAMT). PEAMT activity shows diurnal changes with peak activity at the end of the photoperiod and a decrease overnight. The activity of this enzyme is up-regulated 2 to 3-fold in salt-stressed plants relative to unstressed plants. The objective of this thesis is to determine how PEAMT activity is regulated in vivo. Thus, PEAMT activity, protein and transcript levels were quantified in spinach leaves from plants subjected to different light and salinity conditions. A spinach PEAMT eDNA sequence was used to over-express recombinant PEAMT in the protein expression vector pET30a (+). The presence of a polyhistidine-tag on the overexpressed protein allowed for purification by a cobalt metal affinity column. The affinity purified protein was used to produce polyclonal antibodies for immunoblot hybridization analysis. For these studies, PEAMT protein was first immunoaffinity purified from soluble extracts prepared from leaves and then the protein subjected to electrophoresis by SDS-p AGE. Enzyme assays and immunoblot analysis show PEAMT activity and protein levels increase and become relatively constant in leaves of plants exposed to continuous light. In continuous darkness, PEAMT activity and protein levels decrease and remain low and constant. Thus the pattern of changes in PEAMT activity levels are associated with changes in PEAMT protein levels. In contrast, Northern blot hybridizations show that under conditions of constant light, peamt transcript levels undergo cyclical changes with peak levels at 20 and 40 h and troughs at 28 and 52 h after the continuous light treatment was imposed. These peaks coincide with the dark and light cycles of the normal photoperiod. The same cyclical changes in peamt transcript levels was seen for plants transferred from a normal photoperiod to continuous darkness. Since these changes persist in the absence of a day/night cue we conclude that peamt transcript levels are circadian-regulated. The peamt transcript levels of control unstressed and salt-stressed plants also show circadian rhythms, however the levels found in salt-stressed plants were 0.5 to 2-fold higher than the controls. Therefore, while salinization of plants increases peamt transcript abundance, it does not alter the circadian rhythm that transcripts of this gene display. Changes in PEAMT activity and protein levels are likely controlled by other as yet unknown post-translational mechanisms, processes that override and obscure operation of a circadian rhythm in regulating the level of peamt transcripts. / Thesis / Master of Science (MS)

A fosfoetanolamina é um dos principais constituintes de membranas neuronais de mamíferos e sua deficiência orgânica está intensamente relacionada com epilepsias e distúrbios do sistema nervoso central. Devido a estas evidências, é de suma importância o conhecimento da influência das propriedades físico-químicas, na forma sólida e líquida e suas inter-relações com possível efeito anticonvulsivo. Buscando elucidar algumas destas questões o presente trabalho teve por objetivos avaliar biologicamente os possíveis efeitos anticonvulsivantes e antiepiléticos da fosfoetanolamina sintética FS e FSI, frente a convulsões induzidas por PTZ e nos estados epiléticos, detectados em EEG, de ratos Wistar epiléticos. Foram utilizadas metodologias específicas para ambas as etapas do estudo, incorporando modelos pré-clínicos de avaliações neurológicas de medicamentos pró-anticonvulsivos. Os resultados apontaram efeito na diminuição da intensidade das crises e de óbitos frente ao modelo de indução de crises por PTZ, e efeito anticonvulsivo da forma líquida e sólida frente ao modelo de ratos geneticamente epiléticos, indicando provável ação neurológica da fosfoetanolamina aos eventos biológicos observados. / Phosphoethanolamine is one of mains the mammals membranes constituent and your organic deficiency is intensely related with nervous system central disturbances and epilepsies. Due to this evidences, it belongs to vanish importance knowledge the influence physicist-chemical properties, in the solid form and liquid form and your interrelations with possible effect anticonvulsive. The present work had for goals to evaluate biologically the possible effects anti convulsive and anti epilepticals of synthetical phosphoethanolamine SF and LF, front the convulsions induced for PTZ and in the epileptic state, detected in EEG, of epileptic Wistar mices. They were going used specific methodologies for both the study stages, incorporating models pre-clinical of anticonvulsivements medications neurological evaluations. The results pointed effect in the crises intensity decrease and of deaths front to the crises induction for PTZ, and effect anti convulsive of the liquid and solid form front the model of epileptic genetically mice, indicating phosphoethanolamine probable has neurological action to the observed biological events.

A Fosfoetanolamina Sintética (FO-S), monoester-fosfolípide tem importante papel sobre a proliferação celular, indução da apoptose, em células tumorais, sem, contudo, afetar as células normais. Neste estudo foi avaliado o comportamento biológico e efeitos da toxicidade aguda da fosfoetanolamina sintética (FO-S), em experimentos de dose única e repetidas em camundongos sadios, contribuindo para validação pré-clínica. Os camundongos Balb-c de ambos os sexos receberam o composto FO-S via endovenosa nas doses de 50, 100, 250, 500 e 1000 mg/kg em dose única, e 50, 100 e 250 mg/kg em doses repetidas. No grupo dose única os animais que receberam 500 e 1000 mg/kg de FO-S apresentaram sinais de toxicidade, tais como: mortalidade de 33% dos animais; rebaixamento no sistema nervoso central e autônomo; flutuações das análises hematológicas; aumento dos níveis de TGO e TGP; diminuição de creatinina; análises da medula óssea mostraram diminuição das populações mieloides e linfoides; diminuição de células na fase G0/G1 do ciclo celular, assim como na fase S, e aumento na fase G2/M; alterações histológicas no coração, fígado e rins como necrose, esteatose, hialinização e hiperemia respectivamente. Tanto no grupo dose única, como no grupo doses repetidas, ocorreu aumento no número de reticulócitos no 7º dia após a aplicação, como resposta da medula óssea reativa, e as análises da sua celularidade revelaram atividade positiva do potencial elétrico mitocondrial. No grupo doses repetidas, os animais que receberam 50 mg/kg de FO-S apresentaram no 14º anemia leve, o grupo 100 mg apresentou aumento no número de leucócitos e linfócitos e o grupo 250 mg flutuações nos valores quantitativos de plaquetas, que retornaram à normalidade após 14 dias. As análises da medula óssea revelaram aumento de células no compartimento mieloide no grupo que recebeu 50 mg e aumento da celularidade no compartimento eritroide. A expressão dos marcadores de células precursoras hematopoiéticas da medula óssea, CD34, se mostrou aumentada no grupo que recebeu 250 mg aos 14 dias e diminuição do marcadores de células mielódes e subtipos de linfócitos, CD43, nos grupos que receberam 100 e 250 mg/kg aos 7 e 14 dias. Os animais que receberam 250 mg/kg apresentaram alterações no parênquima pulmonar. A análise de autofagia por citometria de fluxo que não revelou resposta negativa, como estresse oxidativo, apresentando produção normal de vacúolos autofágicos, assim como o teste de presença de micronúcleos não demonstrou danos às células por alterações genéticas induzidas por toxicidade / Synthetic Phosphoethanolamine (FO-S), a monoester phospholipid has an important role on cell proliferation, induction of apoptosis, in tumor cells, without, however, significantly affecting normal cells. In this study the biological behavior and effects of acute toxicity of synthetic phosphoethanolamine (FO-S) were evaluated in single dose and repeated experiments in healthy mice, contributing to the pre-clinical validation of this compound as antitumor phospholipid. Mice of both sexes received intravenous FO-S compound at doses of 50, 100, 250, 500 and 1000 mg / kg in single dose, and 50, 100 and 250 mg / kg in repeated doses. In the single dose group, animals receiving 500 and 1000 mg / kg FO-S showed signs of toxicity, such as: 33% mortality of animals; lowering in the central and autonomic nervous system; fluctuations in hematological analyzes; increased levels of TGO and TGP; decreased creatinine; bone marrow analysis showed decreased myeloid and lymphoid populations; decrease of cells in the G0 / G1 phase of the cell cycle, as well as in the S phase, and increase in the G2 / M phase; histological changes in the heart, liver and kidneys such as hyperemia, necrosis and hyalinization. In both the single dose and repeated dose groups, there was an increase in the number of reticulocytes on the 7th day after application, as a response of the reactive bone marrow, and the cellularity analysis showed positive mitochondrial electrical potential activity. In the repeated dose group, animals receiving 50 mg / kg FO-S presented in the 14th mild anemia, the 100 mg group showed an increase in the number of leukocytes and lymphocytes and the group 250 mg fluctuations in the quantitative platelet values, which returned to the normality at 14 days. Bone marrow analysis revealed increased cell count in the myeloid compartment in the 50 mg group and increased cellularity in the erythroid compartment. Expression of CD34 marrow hematopoietic precursor cell markers was shown to be increased in the 250 mg group at 14 days and a decrease in myelodystic cell markers and CD43 lymphocyte subtypes in the groups receiving 100 and 250 mg / kg at 7 and 14 days. Animals that received 250 mg / kg presented changes in the lung parenchyma. Autophagy analysis was performed by flow cytometry that revealed no negative response, such as oxidative stress, presenting normal production of autophagic vacuoles, as well as the presence of micronuclei test did not demonstrate damage to the cells by genetic changes induced by toxicity