Comparative venomic profiles of three spiders of the genus Phoneutria

Spider venoms induce different physio-pharmacological effects by binding with high affinity on molecular targets, therefore being of biotechnological interest. Some of these toxins, acting on different types of ion channels, have been identified in the venom of spiders of the genus Phoneutria, mainly from P. nigriventer. In spite of the pharmaceutical potential demonstrated by P. nigriventer toxins, there is limited information on molecules from venoms of the same genus, as their toxins remain poorly characterized. Understanding this diversity and clarifying the differences in the mechanisms of action of spider toxins is of great importance for establishing their true biotechnological potential. This prompted us to compare three different venoms of the Phoneutria genus: P. nigriventer (Pn-V), P. eickstedtae (Pe-V) and P. pertyi (Pp-V). Methods: Biochemical and functional comparison of the venoms were carried out by SDS-PAGE, HPLC, mass spectrometry, enzymatic activities and electrophysiological assays (whole-cell patch clamp). Results: The employed approach revealed that all three venoms had an overall similarity in their components, with only minor differences. The presence of a high number of similar proteins was evident, particularly toxins in the mass range of ~6.0 kDa. Hyaluronidase and proteolytic activities were detected in all venoms, in addition to isoforms of the toxins Tx1 and Tx2-6. All Tx1 isoforms blocked Nav1.6 ion currents, with slight differences. Conclusion: Our findings showed that Pn-V, Pe-V and Pp-V are highly similar concerning protein composition and enzymatic activities, containing isoforms of the same toxins sharing high sequence homology, with minor modifications. However, these structural and functional variations are very important for venom diversity. In addition, our findings will contribute to the comprehension of the molecular diversity of the venoms of the other species from Phoneutria genus, exposing their biotechnological potential as a source for searching for new active molecules.(AU)

Development and validation of an LC-MS/MS method for pharmacokinetic study of lobetyolin in rats

Abstract A simple and selective liquid chromatography tandem with mass spectrometry (LC-MS/ MS) method for quantification of lobetyolin in rat plasma was developed and validated. Chromatographic separation was achieved on a Thermo ODS C18 reversed-phase column using 0.1% aqueous formic acid-methanol (50:50, v/v) in an isocratic elution mode at a flow rate of 0.4 mL.min-1. LC/MS performance was done in a positive ion ESI mode and the MS/MS transitions were monitored at m/z 419.3 [M+Na]+ → m/z 203.1 for lobetyolin and m/z 394.9 [M+Na]+ → m/z 231.9 for IS, respectively. The assay exhibited a linear dynamic range over 1.0-500 ng.mL-1 for lobetyolin in plasma. Both the precision (%RSD) and accuracy (RE%) were within acceptable criteria (<15%). Recoveries ranged from 87.0% to 95.6%, and the matrix effects were from 91.0% to 101.3%. After oral administration, the peak plasma concentration of lobetyolin was obtained as 60.1 ng.mL-1 at 1.0 h. The proposed LC-MS/MS method could be applied to a pharmacokinetic study employing 66 samples from 6 Wistar rats

Detecção de esteroides anabólicos androgênicos e seus produtos de biotransformação em dried urine spots por ionização por paper spray acoplada à espectrometria de massas (PS-MS) / Detection of anabolic androgenic steroids and its biotransformation products in dried urine spots by paper spray-mass spectrometry ionization (PS-MS)

Os esteroides anabólicos androgênicos (EAA) são utilizados clinicamente para tratar diferentes doenças, porém propagou-se o uso não terapêutico por atletas de elite e fisiculturistas, com o intuito de aumentar a massa muscular e melhorar o desempenho físico. O uso de substâncias ergogênicas, como fármacos estimulantes e narcóticos analgésicos no esporte, foi proibido em 1967 pelo International Olympic Committee (COI), mas somente em 1976 os EAA entraram para a lista de substâncias proibidas. O uso de EAA está associado a diversos efeitos adversos, principalmente cardiovasculares, neuroendócrinos e distúrbios psiquiátricos, além de dislipidemia, elevação dos marcadores inflamatórios e disfunção endotelial. As análises toxicológicas constam como a maneira mais eficaz de minimizar o doping no esporte. O material é fornecido pelo atleta durante as competições ou treinamentos e previne que os competidores alcancem vantagem competitiva devido ao uso de EAA. A utilização de métodos para amostragem alternativos tem ganhado força, devido à necessidade de técnicas mais práticas que utilizam pouco volume de amostra e possuem facilidade de armazenamento. O dried urine spots é um método no qual pequenas amostras de urina são aplicadas em papéis de filtro para análises qualitativas ou quantitativas. Ele se caracteriza por ser uma técnica rápida, fácil, simples e barata para a coleta, armazenamento e distribuição, além de minimizar os riscos de infecção, podendo ser utilizado na rotina. A técnica de paper spray (PS-MS) foi desenvolvida a partir da relevância de métodos como o dried blood spots por proporcionar análises mais rápidas, apresenta alta especificidade, capacidade de analisar diferentes analitos simultaneamente, baixo limite de detecção e dispensa a necessidade de reagentes específicos. Sendo assim, neste trabalho foi desenvolvido e validado o método de screening de EAAs em dried urine spots por ionização por paper spray acoplada à espectrometria de massas. O método apresentou limites de detecção entre 2-15ng/mL e presença de três interferentes endógenos. Os dez analitos de interesse deste estudo são estáveis por 150 dias em temperatura ambiente. Dessa forma, a análise de EAAs em dried urine spots por PS-MS demonstra grande potencial para se tornar um método alternativo no monitoramento rápido de drogas de abuso

Anabolic androgenic steroids (AAS) are used clinically to treat different diseases, but non-therapeutic use has spread among elite athletes and bodybuilders, with the aim of increase muscle mass and improve physical performance. The International Olympic Committee (IOC) banned the use of ergogenic substances, such as stimulating drugs and analgesic narcotics in sports, in 1967, but only in 1976, AAS were included on the list of prohibited substances. The use of AAS is associated with several adverse effects, mainly cardiovascular, neuroendocrine and psychiatric disorders, in addition to dyslipidemia, elevated inflammatory markers and endothelial dysfunction. Toxicological analyzes are the most effective approach to minimize doping in sport. The material is provided by the athlete during competitions or training and prevents competitors from achieving a competitive advantage due to the use of AAs. The use of alternative sampling methods has gained strength, due to the need for more practical techniques that use low sample volume and can be easily storage. Dried urine spots are a method, which a small amount of urine samples is applied to filter papers for qualitative or quantitative analysis. It is characterized by being a fast, easy, simple and inexpensive technique for collection, storage and distribution, in addition to minimizing the risks of infection, and can be used in the routine. The paper spray technique (PS-MS) was developed based on the relevance of methods such as dried blood spots for providing faster analysis, high specificity, ability to analyze different analytes simultaneously, low detection limit and for eliminating the need for specific reagents. Therefore, this work developed and validated a screening method for AAS in dried urine spots by paper spray-mass spectrometry ionization. The method provided detection limits between 2-15ng/mL and the presence of three endogenous interferents. The ten analytes of interest in this study are stable for 150 days at room temperature. Thus, the analysis of AAS in dried urine spots by PS-MS demonstrates great potential to become an alternative method for the rapid monitoring of drugs of abuse

Avaliação toxicológica da exposição à Cannabis e cocaína na gravidez em cordão umbilical humano: validação de método analítico e prospecção de biomarcadores proteicos de toxicidade / Toxicological assessment of Cannabis and cocaine exposure during pregnancy in human umbilical cord tissue: analytical method validation and prospection of protein biomarkers related to fetotoxicity

O abuso de drogas atinge aproximadamente 35 milhões de pessoas em todo planeta, sendo um problema alarmante em decorrência de graves danos à saúde, como a dependência química e intoxicações fatais. No Brasil, o número de usuários tem crescido principalmente para o consumo de produtos da Cannabis e cocaína, drogas amplamente consumidas, inclusive entre mulheres em período gestacional, trazendo à tona um novo grupo de risco. A exposição gestacional a drogas de abuso está diretamente relacionada a malformações fetais e complicações de saúde para mãe e bebê nos períodos pré- e pós-natal. Tradicionalmente, a avaliação toxicológica da exposição é realizada pela detecção da droga parental e de seus produtos de biotransformação em matrizes materno-fetais por meio de métodos bioanalíticos. Entretanto, estes ensaios não fornecem informações acerca dos impactos fisiológicos ocasionados pela exposição, deixando uma lacuna no que tange às informações sobre os mecanismos e moléculas subjacentes envolvidos em processos de toxicidade. Desse modo, o desenvolvimento de análises toxicológicas mais robustas utilizando tecnologia de ponta, que possam comprovar o uso drogas e também elucidar aspectos de toxicidade é de suma importância, pois auxiliam na compreensão do impacto biológico relativo à exposição humana a xenobióticos. Neste trabalho foram desenvolvidos ensaios bioanalíticos, utilizando o tecido do cordão umbilical para a avaliação da exposição in utero à canabinoides. Foi desenvolvido e validado método QuECheRS adaptado como preparo de amostra, no qual etapas simultâneas de extração e hidrólise alcalina de canabinoides são alcançadas, utilizando cromatografia em fase gasosa acoplada a espectrômetro de massas para detecção de delta-9-tetraidrocanabinol (THC), canabinol (CBN), 11-hidroxi-delta-9-tetraidrocanabinol (11-OHTHC) e 11-nor-9-carboxi-tetrahidrocanabinol (THC-COOH). Também foram desenvolvidas metodologias utilizando LC-MS/MS e Trapped Ion Mobility Mass Spectrometry para análise de proteoma de cordão umbilical humano em diferentes regiões, no intuito de identificar biomarcadores proteicos relativos à fetotoxicidade do uso de drogas na gravidez. Até o presente momento, QuECheRS é utilizado pela primeira vez como abordagem bioanalítica para avaliação de drogas ilícitas em matrizes teciduais materno-fetais e mostrou-se satisfatório para detecção de produtos da Cannabis. Nos ensaios proteômicos, foram identificados potenciais biomarcadores de fetotoxicidade, como as moléculas ACTA 2, Collagen alpha-1 (XVIII), SMC1A, KNL1, KMT2A, em tecidos expostos à Cannabis e/ou cocaína. Tais macromoléculas estão correlacionadas a malformações embriogênicas e complicações de saúde na vida intra-uterina. As metodologias desenvolvidas neste trabalho podem ser úteis para uma melhor avaliação da toxicidade do uso de drogas na gravidez, fornecendo novas pistas sobre a exposição e/ou efeitos tóxicos significativos considerados na avaliação de risco

Drug abuse affects approximately 35 million people worldwide and can be considered a significant burden on society due to severe health problems, e.g. drug addiction and fatal poisonings. In Brazil, the number of users has been growing related to Cannabis and cocaine products, drugs widely used, including among women in gestational period, bringing up a new risk group. Gestational exposure to drugs of abuse is directly related to fetal malformations and health complications for mother and babies in the pre- and postnatal periods. Traditionally, toxicological assessment of exposure is performed by detecting the parent drug and its biotransformation products in maternal-fetal matrices using bioanalytical methods. However, these assays do not provide information about the physiological impacts caused by exposure, leaving a lack of information about the pathways and molecules involved in toxicity processes. Thus, the development of robust toxicological analyzes using cutting-edge technologies in order to prove drug use and also elucidate aspects of toxicity is very important, as they help in understanding the biological impact of human exposure to xenobiotics. Herein, bioanalytical methods using umbilical cord tissue to assess in utero exposure to cannabinoids were developed. A QuECheRS method was developed fully validated as a sample preparation technique for simultaneous extraction and alkaline hydrolysis of cannabinoids, using gas chromatography coupled to mass spectrometry to detect the analytes delta-9-tetrahydrocannabinol (THC), cannabinol (CBN), 11-hydroxydelta-9-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH). LC-MS/MS based proteomics and Trapped Ion Mobility Mass Spectrometry were also developed in order to identify protein biomarkers related to fetotoxicity of drug use in pregnancy. Our works represents the first use of QuECheRS for evaluation of illicit drugs in maternal-fetal tissue and was suitable for detection of Cannabis products. In the proteomic assays, potential biomarkers of fetotoxicity were identified in the exposed tissues, such as ACTA 2, Collagen alpha-1 (XVIII), SMC1A, KNL1, KMT2A. These proteins are related to embryogenic malformations and health complications in intrauterine life. The methodologies developed in this project may be useful for a better assessment of the toxicity of drug use in pregnancy, providing new clues about exposure and/or significant toxic effects that should be considered in the risk assessment

Antiproliferative Effect of Urera baccifera Leaves Against Ovarian Carcinoma Cell Line (OVCAR-3)

Abstract Natural products, especially phytochemicals, have been extensively studies and have exhibited important antiproliferative effects. The American native species Urera baccifera (L.) Gaudich. ex Wedd. (Urticaceae) is widely distributed in Brazil, where it is known as urtiga-vermelha or urtigão. The leaves are popularly used as anti-inflammatory, antirheumatic and in the treatment of gastric disorders. However, the antiproliferative potential of this plant against human tumor cells remain to be elucidated. In this study, we evaluated the antiproliferative effects of U. baccifera leaves extracts and fractions against a panel of human tumor cell lines in vitro besides a chemical evaluation of the most active sample by mass spectrometry (ESI-IT-MSn). The hydroalcoholic extract was inactive while dichloromethane extract showed moderate cytostatic activity against ovarian carcinoma cell line (OVCAR-3, GI50 = 1.5 μg/mL). More, the ethyl acetate and n-butanol fractions did not show important activity against tumour cell while the dichloromethane and hexane fractions showed moderate cytostatic activity against ovarian tumor cell line (OVCAR-3, GI50 = 12.7 and 9.4 μg/mL, respectively). Finally, the chemical profile evaluated by mass spectrometry (ESI-IT-MSn) allowed the detection of flavonoids in the HEU and hydroxylated fatty acid in DEU that can explain partially the biological effects observed. This is the first report of the antiproliferative effects of U. baccifera, and DEU has shown potential as a promising source of bioactive compounds.

Determinação de estimulantes, esteroides anabolizantes, diuréticos e laxante por HPLC-DAD e LC-MS/MS em suplementos alimentares adulterados disponíveis no Brasil / Determination of stimulants, anabolic steroids, diuretics and laxative by HPLC-DAD and LC-MS/MS in adulterated dietary supplements available in Brazil

A adulteração de suplementos alimentares pela adição de substâncias farmacologicamente ativas e um problema que vem se agravando nos últimos anos. Essas substâncias são adicionadas intencionalmente nos produtos, com objetivo de melhorar a sua eficácia, sem que essa adição seja devidamente informada nos rótulos. O consumo de suplementos adicionados de substâncias farmacologicamente ativas nao declaras nos rótulos e, alem de um problema de saúde pública, um risco a carreira de atletas profissionais, quando se trata de doping no esporte. Neste trabalho foram desenvolvidos métodos analíticos baseados em cromatografia líquida para detecção de 19 substâncias farmacologicamente ativas em amostras de suplementos alimentares, nos niveis de contaminação cruzada e adulteração. Para tal, empregou-se a cromatografia líquida de alta eficiência acoplada a detector de arranjo de diodos (HPLC-DAD) e a cromatografia líquida acoplada a espectrometria de massas sequencial (LC-MS/MS). As substâncias investigadas apresentam ação androgênica e anabólica (i.e. testosterona, metiltestostetona, propionato de testosterona, decanoato de testosterona, trembolona, estanozolol, dehidroepiandrosterona, androstenediona, decanoato de nandrolona, oxandrolona e metasterona), estimulante (i.e. cafeína), anorexigena (i.e. sibutramina), diurética (i.e. amilorida, bumetanida, furosemida, hidroclorotiazida e clortalidona) e laxante (i.e. fenolftaleina). Entre os métodos de preparo de amostra avaliados (Quechers e extração sólido-líquido seguida de uma etapa de precipitação de proteína), a extração sólido-líquido empregando metanol como solvente extrator e ZnSO4 como agente precipitante apresentou-recuperação acima de 80% para todas as substâncias avaliadas, sendo selecionado para o fim deste estudo. Os métodos analíticos propostos apresentaram limites de detecção e de quantificação na faixa de contaminação, foram seletivos e lineares com r2 superior a 0,99 na faixa de concentração de interesse para todas as substâncias e valores de recuperação, precisão e exatidão dentro dos valores aceitáveis. Um conjunto amostral representativo dos suplementos alimentares comercializados no Brasil, constituído por 230 amostras, foi analisado sendo que mais de 25% do conjunto amostra foram positivos para cafeína (48), sibutramina (14), fenolftaleína (2) e furosemida (3), isoladas ou associadas entre si. Os métodos desenvolvidos utilizaram um preparo de amostra simples e apresentaram resultados satisfatórios para a investigação de possível adulteração ou contaminação com 19 das substâncias de interesse. Dos 58 suplementos alimentares adulterados, apenas 11 podem ser considerados adulterados por contaminação cruzada. Os demais, são consideradas adições dolosas por parte dos fabricantes com objetivo de melhorar a eficiência dos seus produtos. Os resultados aqui apresentados indicam a necessidade de ações mais efetivas por parte das autoridades sanitárias no sentido de fiscalizar com mais eficiência a produção e a comercialização desses produtos e alertar a população para que fiquem atentos a possível adulteração de suplementos alimentares e aos riscos associados ao consumo desses produtos

The adulteration of dietary supplements by the addition of pharmacologically active substances is a serious issue, which is aggravating steadily. These substances are added intentionally in various products, with the aim of improving their effectiveness, but without proper labeling stating so. In addition to a public health problem, the consumption ofsupplements added with undeclared pharmacologically active substances also represents a career risk for professional athletes when it comes to doping in sport. In the present work, analytical methods based on liquid chromatography were developed for the detection of 19 pharmacologically active substances in dietary supplement samples at the levels of crosscontamination and adulteration. For this purpose, high performance liquid chromatography/diode array detector (HPLC-DAD) and liquid chromatography/tandem mass spectrometry (LC-MS/MS) were used. The investigated substances comprise of androgenic and anabolic effects (ie testosterone, testosterone propionate, testosterone decanoate, trenbolone, stanozolol, dehydroepiandrosterone, androstenedione, nandrolone decanoate, oxandrolone and metasterone), stimulant (ie caffeine), anorexigenic (ie sibutramine), diuretic (ie amiloride, bumetanide, furosemide, hydrochlorothiazide and chlorthalidone) and laxative (ie phenolphthalein). Among the sample preparation methods evaluated (Quechers and solidliquid extraction followed by a protein precipitation step), solid-liquid extraction using methanol as extraction solvent and ZnSO4 as the precipitating agent has been chosen as it has shown recovery values above 80% for all evaluated substances. The proposed analytical methods had limits of detection and quantification within the contamination range, they were also selective and linear showing r2 values higher than 0.99 in the concentration range of interest for all substances and accuracy within acceptable values. A representative sampling of dietary supplements marketed in Brazil, consisting of 230 samples, was analyzed and more than 25% have shown to be positive for caffeine (48), sibutramine (14), phenolphthalein (2) and furosemide (3), isolated or associated with each other. The methods developed used a simple sample preparation and presented satisfactory results for the investigation of possible adulteration or cross-contamination for the 19 of the substances of interest. From a total of 58 adulterated dietary supplements, only 11 could be considered adulterated by crosscontamination. The remaining are considered to be intentional additions by manufacturers in order to improve the efficiency of their products. The results presented in this study indicate the need for more effective measures by the health authorities towards the production and marketing of these products so that the general public is aware of their potential adulteration and the risks associated with their consumption

Exploring the role of lipids in protein modification and amyotrophic lateral sclerosis / Explorando o papel dos lipídeos na modificação de proteínas e esclerose lateral amiotrófica

Lipids are a diverse and ubiquitous group of compounds, which have several biological functions such as structural components of cell membranes, energy storage, and participation in signaling pathways. Free radicals or reactive oxygen species could attack polyunsaturated fatty acid esterified to phospholipids generating oxidized products. Once oxidized, lipids are able to modify amino acids residues in proteins leading to modulation signaling pathways and cellular redox balance. Furthermore, alteration of lipid homeostasis is also linked to development and progression of neurodegenerative diseases. The purposes of this study were (i) to investigate the role of lipids in protein aggregation, (ii) to investigate the plasma lipidome of an ALS rat model (SOD1G93A rats), and (iii) to investigate the effect of high-fat diet in plasma lipidome of an ALS rat model. In chapters 1 and 2, the interaction between cytochrome c (cytc) and cardiolipin hydroperoxide (CLOOH), as well as cholesterol hydroperoxide (ChOOH) promoted protein aggregation. Mass spectrometry analysis of tryptic peptides from CLOOH-containing reaction revealed K72 and H26 consistently modified by 4- hydroxynonenal (4-HNE). Further, adduction of K27, K73 and K88 were detected with 4- oxynonenal (4-ONE). For the first time, we characterized the dityrosine cross-linked peptides at Y48-Y74, Y48-97 and Y74-Y97 in oligomeric cytc. Similarly, ChOOH-containing reaction showed dityrosine cross-linked peptides at Y48-Y48, Y48-Y74 and Y48-Y97 in dimeric cytc. In accordance to previous studies, the proposed mechanism under covalent protein oligomerization mediated by lipid hydroperoxide could be related to modification of lysine and tyrosine residues. In chapter 3, we characterized the lipid composition of blood plasma in amyotrophic lateral sclerosis (ALS), since dysregulation of lipid metabolism is increasingly associated with neuropathology. Using untargeted lipidomics approach based on liquid chromatography coupled to mass spectrometry, we found main alterations in triglycerides, phospholipids and sphingolipids in symptomatic ALS rats relative to controls. Additionally, for the first time we reported acylceramides species in the plasma. In order to investigate the source of these lipid alterations, we analyzed the lipid content of fractioned lipoproteins. Triglycerides and phospholipids were found in very low-density lipoprotein (VLDL), while acylceramides and hexosylceramides were found enriched in high-density lipoprotein (HDL). In chapter 4, high-fat diet containing lard or high-fish oil as much as 60% of total lipids has both the largest change on plasma lipid composition. Overall survival was not statistically different when compared to control diet. Increased levels of acylceramides, hexosylceramides and acylcarnitines were observed in ALS rats fed a control diet or high-fat diet in comparison to WT controls. Importantly, untargeted lipidomic analysis of blood plasma highlighted acylceramide d18:1/24:1+20:4 as potential biomarkers of ALS progression. Thus, our lipidomic analysis provides a novel insight into the molecular level event driving molecular dysregulation in ALS. Additional research is needed to determine the effect of plasma lipid alteration on motor neuron process and energetic metabolism. Collectively, our findings reinforce the idea that lipids play a relevant role in modulating cellular processes linked to protein aggregation and neurodegeneration

Os lipídeos são moléculas que possuem várias funções biológicas importantes, atuando como componente de membranas celulares, servindo com fonte de reserva de energia e participando de vias de sinalização. Os ácidos graxos poli-insaturados esterificados aos fosfolipídeos, por exemplo, são potenciais alvos para o ataque de radicais livres gerando produtos oxidados que são capazes de modificar resíduos de aminoácidos em proteínas levando a modulação das vias de sinalização e balanço redox. Por outro lado, alteração na homeostase do metabolismo dos lipídeos está relacionada ao desenvolvimento e progressão de doenças neurodegenerativas. Tendo em vista a importância dos lipídeos nos processos biológicos, os objetivos desse estudo foram (i) investigar o papel dos lipídeos na agregação proteica (capítulo 1 e 2), (ii) investigar as alterações na composição lipídica do plasma de rato modelo SOD1G93A de esclerose lateral amiotrófica (ELA) (capítulo 3) e (iii) investigar o efeito da suplementação de dietas hiperlipídicas na composição lipídica do plasma de rato modelo SOD1G93A (capítulo 4). No capítulo 1 e 2, a interação do citocromo c (citc) com hidroperóxido de cardiolipina (CLOOH) e hidroperóxido de colesterol (ChOOH) promove a agregação covalente do citc. Análise por nLC-MS/MS dos peptídeos digeridos identificou resíduos de lisina (K72) e histidina (H26) modificado por 4-hidroxininenal (4-HNE), enquanto os resíduos K27, K73 e K88 foram modificados por 4-oxinonenal (4-ONE). Pela primeira vez, nós caracterizamos ditirosinas (Y48-Y74, Y48-97 e Y74-Y97) na reação do citc com CLOOH. Também foram caracterizadas ditirosinas envolvendo os resíduos Y48-Y48, Y48-Y74 e Y48-Y97 na reação com ChOOH. Esses resultados corroboram com estudos anteriores que sugerem um mecanismo de agregação proteica envolvendo a perda da carga positiva de lisina e formação de ditirosina pela combinação de radicais de tirosil. No capítulo 3, a análise da composição lipídica do plasma de ratos SOD1G93A utilizando LC-MS/MS revelou alterações significativas na composição de triglicérides, glicerofosfolipídeos e esfingolipídeos em ratos sintomáticos comparado com os assintomáticos. É importante destacar que pela primeira vez acilceramidas foram identificadas em plasma de rato modelo para ALS. Análise da composição lipídica de lipoproteínas isoladas, maior fonte de lipídeos circulantes no plasma, mostraram alterações de triglicérides e glicerofosfolipídeos em VLDL. As acilceramidas e as hexosilceramidas, por sua vez, foram encontradas em maior abundância em HDL. No capítulo 4, a suplementação com dietas hiperlipídicas (rica em banha de porco e óleo de peixe) alterou significativamente o perfil lipídico do plasma em relação a doença. Contudo, não foi observado aumento significativo na sobrevida dos ratos ALS comparado com dieta controle. Independente da dieta, a concentração plasmática de acilcarnitina, hexosilceramidas e acilceramidas foram significativamente aumentadas em ratos ALS comparado com WT. A análise do perfil lipídico do plasma mostrou que a acilceramida d18:1/24:1+20:4 pode ser um potencial marcador de progressão da ALS. Dessa forma, os resultados mostrados fornecem uma visão enriquecedora sobre o evento a nível molecular que conduz a desregulação lipídica na ELA. Coletivamente, nossos resultados reforçam a importância dos lipídeos na modulação dos processos celulares ligados a agregação de proteínas e na neurodegeneração

Guideline values and human risk assessment for the presence of anti-inflammatory drugs remaining in drinking water after lab scale treatment

This study aimed to determine whether the anti-inflammatory drugs that are most commonly consumed in Brazil, including diclofenac, ketoprofen, naproxen, indomethacin, ibuprofen and acetaminophen, are present in drinking water and to derive guideline values to characterize the human risk. These pharmaceuticals were quantified in surface waters by LC-MS/MS with solid phase extraction, both before and after conventional treatment on a laboratory scale, using a jar test assay. The methods used to quantify these drugs showed good results: the chromatographic analysis obtained correlation coefficients between 0.9952 and 0.9991, with limits of quantification of 0.5 ng.mL-1 - 50 ng.mL-1 and precision standard deviations (0.08 - 2.08). Only ketoprofen and ibuprofen were not completely removed through the jar test. Environmental samples were collected and handled by the same method; the values |for ketoprofen and ibuprofen after treatment were 18.67 - 19.65 ng.L-1 (±17%) and 166.70 - 244.73 ng.L-1 (±14%), respectively. Human risk was assessed by comparing the guideline values for each compound to the concentrations obtained in the environmental samples, considering the toxicological backgrounds, following WHO (2011) method. The results suggest that the concentrations of ketoprofen and ibuprofen found in drinking water do not pose a risk to human health, even with chronic consumption

Síntese e estudos espectroscópicos de organometálicos de rutênio-areno contendo fármacos anti-inflamatórios e derivados como ligantes / Synthesis and spectroscopic studies of ruthenium-arene organometallics bearing anti-inflammatory drugs and derivatives as ligands

Compostos organometálicos do tipo rutênio-areno têm sido estudados no transcurso dos últimos anos em razão do potencial que apresentam para o tratamento de doenças dentre as quais se destaca o câncer. Neste contexto, o presente trabalho teve como principal objetivo o estudo de organometálicos de Ru(II)-p-cimeno contendo como ligantes fármacos anti-inflamatórios não esteroides (FAINEs) ou seus derivados piridinaamida (FAINE-amida). Foram realizadas as sínteses de duas classes de compostos de fórmulas gerais [RuCl(p-cimeno)L] e [RuCl2(p-cimeno)Lam] em que L = ibuprofeno, naproxeno ou indometacina e Lam = derivado amida desses FAINES, respectivamente. A composição e estrutura dos compostos foram elucidadas principalmente com base em análise elementar, espectrometria de massas (ESI-MS), espectroscopia de ressonância magnética nuclear (1H RMN, 13C RMN, HSQC, HMBC) e espectroscopia vibracional ATR/FT-IR. Os dados indicaram que todos os fármacos-ligantes utilizados estabilizam a unidade Ru(II)-areno, sendo que os carboxilatos coordenam-se ao Ru(II) de modo bidentado por ambos os átomos de oxigênio, enquanto que a coordenação dos derivados amida ocorre pelo nitrogênio do anel piridínico. No entanto, em contraste ao comportamento em solventes não-coordenantes como clorofórmio, estudos em solução indicaram que a presença de dimetilsulfóxido promove dissociação do fármaco ligante acompanhada pela coordenação do solvente (gradual, no caso de L, ou imediata total no caso de Lam). Resultados preliminares de estudos de espectroscopia de fluorescência sugerem interação dos compostos de Ru(II)-areno-indometacina com albumina de soro humano (HSA)

Ruthenium-arene organometallics have been investigated in recent years due to the potential for treatment of diseases among which cancer is highlighted. In this context, the main objective of the present work is the study of organometallics of Ru(II)-p-cymene bearing non steroidal anti-inflammatory drugs (NSAIDs) or their pyridine-amide (NSAIDamide) as ligands. Two classes of compounds of general formula [RuCl(p-cymene)L] and [RuCl2(p-cymene)Lam], in which L = ibuprofen, naproxen or indomethacin and Lam = amide derivative of these NSAIDs, respectively have been synthesized. The composition and the structure of these compounds have been elucidated mainly based on elemental analysis, mass spectrometry (ESI-MS), nuclear magnetic resonance spectroscopy (1H RMN, 13C RMN, HSQC, HMBC) and vibrational spectroscopy (ATR/FT-IR). The data indicate that all the used drug-ligands stabilize the Ru(II)-arene framework, being that the carboxylates coordinate Ru(II) in bidentate mode through both oxygen atoms while the coordination of the amide derivatives occurs via nitrogen atom of the pyridine ring. However, in contrast to the behavior in non-coordinating solvents such as chloroform, studies in solution indicate that the presence of dimethylsulfoxide promotes dissociation of the drug ligand accompanied by the coordination of the solvent (gradual, for L, or total immediate for Lam). Preliminary results from fluorescence spectroscopy suggest interaction of the Ru(II)-arene-indomethacin compounds with human serum albumin (HSA)

A análise proteômica no contexto forense / The proteomic analysis in the forensic context

Realizou-se uma revisão de literatura consultando as bases de dados Medline, LILACS, BBO e Scielo, de 1997 a 2017, com o objetivo de ressaltar a importância do estudo da proteômica e sua aplicação nas diversas áreas forenses. Conclui-se que a evolução da tecnologia de espectrometria de massa juntamente com os bancos de dados proteômicos aplicados para o estudo das proteínas do corpo humano são de grande importância no contexto forense e podem fornecer subsídios nas diversas áreas da Criminalística, assim como na identificação e diferenciação de fluidos, tecidos e órgãos do corpo humano; na Criminologia no diagnóstico de doenças mentais; na Arqueologia e na Antropologia Forense no estudo da evolução das espécies, na estimativa da ancestralidade, do sexo, da cor dos olhos e cabelo e da idade, auxiliando nos processos de identificação humana.

It was performed a literature review on Medline, LILACS, BBO and Scielo databases from 1997 to 2012 in order to highlight the importance of the study of proteomics and its application in the various forensic areas. It is concluded that the evolution of the technology of mass spectrometry together with the proteomic databases applied to the study of proteins of the human body are of great importance in the forensic context and can provide subsidies in the different areas of Criminalistics as well as in the identification and differentiation of fluids, tissues and organs of the human body; in Criminology in the diagnosis of mental illness; in Archeology and Forensic Anthropology in the study of the evolution of species, in the estimation of ancestry, sex, eye color and hair and age, aiding in the processes of human identification.

Estudo proteômico de variedades de milho ( Zea mays L. ) obtidas por melhoramento clássico e por recombinação genética / Proteomic study of maize ( Zea mays L. ) varieties obtained by classical breeding and genetic recombination.

O melhoramento genético clássico de sementes milho (Zea mays L.) permitiu desenvolver inúmeras variedades, incluindo o milho com qualidade proteica melhorada (Quality Protein Maize, QPM), que visava aumentar os teores proteicos e as propriedades nutricionais. Por outro lado, novas variedades comerciais foram obtidas por vegetais geneticamente modificados (GM), com foco em parâmetros agronômicos. Em ambos os casos, a segurança dessas variedades para uso como alimento é uma das principais preocupações dos desenvolvedores e dos órgãos de regulamentação. A Equivalência Substancial é a base do sistema de avaliação da segurança de culturas geneticamente modificadas, no entanto alterações na expressão de proteínas não são devidamente analisadas e esclarecidas. As abordagens proteômicas complementam as técnicas de avaliação de biossegurança para alimentos GM, bem como permitem investigar possíveis efeitos indesejáveis derivados do melhoramento clássico. Os objetivos do presente estudo foram caracterizar e comparar os perfis proteicos de variedades de milhos convencionais melhorados (QPM) e geneticamente modificados (GMs), contra suas respectivas linhas convencionais utilizando técnicas proteômicas como eletroforese bidimensional (2-DE) e bottom up shotgun (gel-free). Num primeiro estudo, foram utilizadas três amostras de milho, sendo duas variedades convencionais com QPM (QP1 e QP2) e uma variedade convencional normal (CN). No segundo estudo, foram analisadas duas cultivares de milho GM (GM1 e GM2) e seus respectivos convencionais genitores (CG1 e CG2). As composições químicas de todas as amostras também foram avaliadas quanto a Equivalência Substancial. O extrato bruto proteico foi submetido à análise de eletroforese unidimensional (1-DE), bidimensional (2-DE) e bottom up shotgun (gel-free). As imagens dos mapas proteicos foram analisadas pelo software Image Master 2D Platinum 7.0 (GE). Os spots diferencialmente expressos e selecionados foram sequenciados por MS. Pela composição química das principais frações das amostras de milho foi possível identificar a equivalência substancial entre as amostras convencionais e GMs, bem como QPMs e sua convencional dentro das faixas de variabilidade esperadas da espécie. Nos géis 1-DE foram observadas bandas proteicas com perfis similares entre os grupos de amostras avaliadas para ambos estudos. Nas imagens dos géis 2-DE não houveram alterações extremas entre as amostras de milhos GMs e seus respectivos convencionais genitores (CGs), mas apenas diferenças na intensidade dos spots proteicos. As variedades QPMs e CN apresentaram diferenças devido à distribuição dos spots. Os mapas proteicos das amostras CG1 x GM1 e CG2 x GM2 apresentaram maior semelhança com porcentagens de matchings superiores a 70 %, enquanto as porcentagens de matchings entre variedades diferentes (QPMs e CN) foram menores. No total foram identificadas 219 proteínas das amostras CGs x GMs e QPMs x CN, classificadas quanto aos seus processos biológicos e função molecular. Em conclusão, foram encontradas diferenças entre os cultivares GMs e CGs, indicando uma variação normal entre variedades de milho, que não comprometem a segurança alimentar das amostras estudadas. Quanto às amostras com QPM e CN as diferenças encontradas são devido à sua distância nas linhagens ou germoplasma

The classic genetic breeding of corn seeds (Zea mays) has enabled the development of many varieties, including corn with improved protein quality (Quality Protein Maize, QPM), which aimed to increase protein levels and nutritional properties. On the other hand, new commercial varieties have been obtained out of genetically modified (GM) vegetables, with a focus in agronomic parameters. In both cases, the safety of these varieties for food use is one of the main concerns for the developers and for the regulatory agencies. Substantial Equivalence is the basis of the safety evaluation system for genetically modified crops, however, alterations in the protein expressions are not been properly analyzed and clarified. The protein approaches complement the techniques of biosafety evaluation for GM foods, as well as allow for possible undesirable effects derived from classic improvement to be investigated. The goals of the current studies were to characterize and compare the protein profiles of the different varieties of conventionally improved (QPM) and genetically modified (GM) corn, against their respective conventional lines using proteomic techniques, such as, two-dimensional electrophoresis (2-DE), bottom up shotgun (gel-free) and masses spectrometry (MS). In a first instance of the study, three samples of corn were used, two of conventional varieties with QPM (QP1 and QP2) and one conventional normal variety (CN). In a second instance of the study, two cultures of GM corn (GM1 and GM2) were analyzed and their respective conventional genitors (CG1 and CG2). The chemical compositions of all the samples were also evaluated for their Substantial Equivalence. The protein raw extract was submitted to analysis of one-dimensional (1-DE), two-dimensional (2-DE) electrophoresis, and bottom up shotgun (gel-free). The protein image maps were analyzed by the Image Master 2D Platinum 7.0 (GE) software. The spots which were expressed and selected differentially were sequenced by MS. By the chemical composition of the main fractions of the samples of corn, it was possible to identify the substantial equivalence between the conventional samples and GMs, likewise with OPMs and their conventional in the ranges of variability which were expected for the species. On the 1-DE gel, it was observed protein bands with similar profiles amongst the groups of evaluated samples for both studies. In the images of the 2-DE gel, there were no alterations between the GM corn and their respective conventional genitors (CGs), but only differences in intensity of the protein spots. The OPM and CN varieties presented differences due to the distribution of the spots. The protein maps of samples CG1 vs. GM1 and CG2 vs. GM2 presented greater similarities with the percentages of matchings superior to 70%, while the percentage of matchings among different varieties (QPMs and CN) were smaller. In total, there were 219 proteins identified in the samples CGs vs. GMs and QPMs vs. CN, classified by the biologic processes and molecular function. In conclusion, there were found differences between the cultures of GMs and CGs, indicating a normal variation among the corn varieties, which do not affect the food security of the studied samples. As per the samples with QPM and CN, the differences found were due to the line distances or germplasm

Differential toxicological endpoints of di(2-ethylhexyl) phthalate (DEHP) exposure in MCF-7 and MDA-MB-231 cell lines: Possible estrogen receptor α (ERα) independent modulations.

Wide spread use of Di-(2-ethylhexyl) phthalate (DEHP) has made it a ubiquitous contaminant in today’s environment, responsible for possible carcinogenic and endocrine disrupting effects. In the present investigation an integrative toxico-proteomic approach was made to study the estrogenic potential of DEHP. In vitro experiments carried out with DEHP (0.1-100 μM) induced proliferations (E-screen assay) in human estrogen receptors-α (ERα) positive MCF-7 and ERα negative MDA-MB-231 breast cancer cells irrespective of their ERα status. Further, DEHP suppressed tamoxifen (a potent anti-breast cancer drug) induced apoptosis in both cell types as shown by flowcytometric cell cycle analysis. Label-free quantitative proteomics analysis of the cell secretome of both the cell lines indicated a wide array of stress related, structural and receptor binding proteins that were affected due to DEHP exposure. The secretome of DEHP treated MCF-7 cells revealed the down regulation of lactotransferrin, an ERα responsive iron transport protein. The results indicated that toxicological effects of DEHP did not follow an ERα signaling pathway. However, the differential effects in MCF-7 and MDA-MB-231 cell lines indicate that ERα might have an indirect modulating effect on DEHP induced toxicity.

Characterization and anti-staphylococcal activity of the essential oil from Turnera subulata Sm

The present study is pioneer in analyzing the chemical composition of the essential oil from aerial parts of Turnera subulata Sm. and evaluates their antibacterial activity against a panel of drug-resistant strains of Staphylococcus aureus. The aerial parts were kiln-dried and then powdered in mechanical mill, and was subjected to hydrodistillation in a Clevenger-type apparatus. The components were analyzed using a mass spectrometrycoupled gas chromatography (GC-MS), and the identification of substances was performed by comparison of the mass spectra obtained with the mass spectra of the database of the GC-MS and retention indices . The essential oil was evaluated using the method of broth dilution at concentrations ranging from 3.125µg/mL to 3200µg/mL. It was possible to identify 45 substances (92.1%) of the essential oils, and the major components were trans-caryophyllene (6.7%), citronellol (5.6%), sphatulenol (5.3%), α-cadinol (4.3%), n-tricosano (4.3%), geraniol (4.1%) and trans-geranilacetone (3.7%), n-pentacosano (3.5%), globulol (3.4%), caryophyllene oxide (3.2%). The essential oil of T. subulata Sm. showed effective antibacterial activity for the various strains of S. aureus tested, with the MIC values between 25 µg/mL and 1600 µg/mL. The study of the essential oil of Turnera subulata Sm. showed that it consists of a complex mixture of several classes of compounds having the sesquiterpenes as major constituents followed by monoterpenes, and showed an antibacterial activity significant front the strains tested.

O presente estudo é pioneiro em analisar a composição química dos óleos essenciais das partes aéreas de Turnera subulata Sm. e sua atividade antibacteriana frente à Staphylococcus aureus resistentes a antibióticos. As partes aéreas da planta foram secas em estufa, pulverizadas em moinho mecânico e submetidas à hidrodestilaçao em aparato tipo Clevenger. A composição dos óleos essenciais foi analisada por cromatografia gasosa acoplada a espectrometria de massas (CG-EM), e a identificação realizada por comparação dos espectros de massas com a biblioteca do CG-EM e índices de retenção. A atividade antimicrobiana do óleo essencial foi avaliada usando o método de diluição em caldo, em concentrações que variaram de 3.125µg/mL to 3200µg/mL. Foi possível identificar 45 substâncias (92,1%) do óleo essencial, sendo os componentes majoritários: trans-cariofileno (6,7%), citronelol (5,6%), espatulenol (5,3%), α-cadinol (4,3%), n-tricosano (4,3%), geraniol (4,1%) e trans-geranilacetona (3,7%), n-pentacosano (3,5%), globulol (3,4%) e óxido de cariofileno (3,2%). O óleo essencial de T. subulata Sm. mostrou atividade antibacteriana eficaz para as várias cepas de S. aureus testadas, com valores de CIM entre 25 µg/mL e 1600 µg/mL. O estudo do óleo essencial de Turnera subulata Sm. evidenciou sua complexa mistura, contendo várias classes de substâncias, tendo os sesquiterpenos como constituintes majoritários seguido dos monoterpenos, e mostrou significativa atividade antibacteriana frente as cepas testadas.

Structure investigation of piroxicam and tenoxicam using thermal analyses, mass spectrometry and semi-empirical MO calculations

Steroidal and non-steroidal anti-inflammatory agents such as oxicam group [e.g. piroxicam [Pir] and tenoxicam [Ten] were investigated using thermal analyses [TA] measurements [TGA, DTGA, DTA and DDTA] in comparison with El mass spectral [MS] fragmentation at 70 eV. Semi-emperical molecular orbital [MO] calculations have been carried out using PM3 described by Stewart on Pir and Ten both as neutral molecules and the corresponding positively charged molecular ions. These calculations included molecular geometries such as bond length, bond order, bond strain, atomic charge distribution and hybridization, and heat of formation of these drugs. Thermal analyses reveal a high response of Pir and Ten to temperature variation with a cleavage of six bonds in aliphatic side chains around O, S, and N heteroatom, leaving aromatic radicals as final products, which become volatile at high temperature. TA mostly involved fragmentation of SO2 gas molecule at first followed by C, N, and O containing fragments as CH3CN, CHO, NHCONHCO, and NHCO. The MS fragmentations indicate the presence of the same final products as given by TA technique. Mass fragmentation pathways refer to the loss of more or less the same fragments via cleavage of seven bonds of Pir and nine bonds of Ten molecular ions, in parallel and many consecutive steps as explained by geometries values calculated by MOC. These may refer to the instability of Pir and Ten molecular ions in comparison with their neutral forms. This instability is explained by comparison of the calculated partial charges on their atoms and/or their heat of formation values. The best pathway in both TA and MS techniques is that starts with the loss of SO2 gas molecule and followed by the loss of HCO and CH3CN molecules. The partial charge values and the stereo structures of ionic and neutral form refer to a distinct phenomenon of collection of voluminous charge density on SO2 group covering in space most atoms behind. It is explained by the highest electron withdrawing abilities of the heteroatom, two O and S in this group as a result of their high electro negativities. This rationalized the starting of fragmentation process in TA and MS with the loss of SO2 gas molecules. Therefore, a MOC was applied to declare both TA and MS observations