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1.
Braz. j. biol ; 83: 1-9, 2023. tab, graf, ilus
Article in English | LILACS, VETINDEX | ID: biblio-1468896

ABSTRACT

Previous studies have suggested that arsenic crosses the placenta and affects the fetus development. The study under consideration aims to show comparative ameliorative effect of Moringa oleifera leaf and flower extracts against sodium arsenate induced fetus toxicity of mice. Pregnant mice (N=44) were kept in lab and divided into eleven group from (A to K) and were orally administered the doses 6 mg/kg, 12 mg/kg for sodium arsenate, 150 mg/kg and 300 mg/kg for Moringa oleifera leaf extracts (MOLE) and 150 mg/kg and 300 mg/kg for Moringa oleifera flower extracts (MOFE) comparing with control. The investigation revealed evident reduction in the fetuses weight, hind limb, fore limb, tail and snout length, crown rump and head circumferences well as malformations in tail, feet, arms, legs, skin and eyes in the negative control group (only administered with sodium arsenate). Co-administration of sodium arsenate with MOLE and MOFE ameliorate the reversed effect of sodium arsenate on the shape, length, body weight and DNA damage of fetus significantly at 95% confidence interval. However, Moringa oleifera leaf extract showed more significant results in comparison to Moringa oleifera flower extract. Hence concluded that Moringa oleifera leaf extract ameliorated the embryo toxic effects of sodium arsenate and can be used against environmental teratogens.


Estudos anteriores sugeriram que o arsênio atravessa a placenta e afeta o desenvolvimento do feto. O estudo em consideração visa mostrar o efeito melhorador comparativo de extratos de folhas e flores de Moringa oleifera contra a toxicidade fetal induzida por arseniato de sódio em camundongos. Camundongos grávidas (N = 44) foram mantidos em laboratório e divididos em 11 grupos (de A a K) e foram administrados por via oral nas doses de 6 mg/kg, 12 mg/kg para arseniato de sódio, 150 mg/kg e 300 mg/kg para extratos de folhas de Moringa oleifera (MOLE) e 150 mg/kg e 300 mg/kg para extratos de flores de Moringa oleifera (MOFE) em comparação com o controle. A investigação revelou redução evidente no peso do feto, membro posterior, membro anterior, comprimento da cauda e focinho, coroa, nádega e circunferência da cabeça, bem como malformações na cauda, pés, braços, pernas, pele e olhos no grupo de controle negativo (apenas administrado com arseniato de sódio). A coadministração de arseniato de sódio com MOLE e MOFE melhora significativamente o efeito reverso do arseniato de sódio na forma, comprimento, peso corporal e dano ao DNA do feto, com intervalo de confiança de 95%. No entanto, o extrato da folha da Moringa oleifera apresentou resultados mais significativos em comparação ao extrato da flor da Moringa oleifera. Portanto, concluiu que o extrato da folha de Moringa oleifera melhorou os efeitos tóxicos do arseniato de sódio para o embrião e pode ser usado contra teratógenos ambientais.


Subject(s)
Female , Animals , Pregnancy , Mice , Arsenates/toxicity , Comet Assay/veterinary , Fetus/abnormalities , Fetus/drug effects , Prenatal Injuries/veterinary , Moringa oleifera/embryology
2.
An. Fac. Cienc. Méd. (Asunción) ; 55(1): 27-38, 20220401.
Article in Spanish | LILACS, BDNPAR | ID: biblio-1366663

ABSTRACT

Introducción: Procesos como la mutagénesis, la carcinogénesis y la teratogénesis son producto de la interacción de agentes de origen endógeno como exógeno que interactúan con la molécula de ADN en forma crónica produciendo rupturas en la doble hélice, y en cromosomas completos resultando en la inestabilidad genómica. El estrés oxidativo al que se encuentran sometidas las células al formarse las especies reactivas de oxígeno (ROS) y también las especies reactivas de nitrógeno (RNS), que pueden provenir de radicales producidos a consecuencia de la diabetes o en estados iniciales de la enfermedad renal crónica o como respuesta a procesos inflamatorios en estados avanzados de estas patologías, actúan como agentes genotóxicos endógenos.Objetivos: Esta investigación tuvo como objetivo determinar el daño basal en la molécula de ADN de pacientes diabéticos hemodializados, a través del ensayo del Cometa, como un bioindicador de inestabilidad genómica., durante seis meses de tratamiento. Materiales y métodos: Se planteó un estudio longitudinal prospectivo de cohorte para comparar los diferentes niveles de daño antes y durante los primeros seis del tratamiento de hemodiálisis. Se evaluó con el test del cometa o electroforesis de células individuales, el daño basal en muestras de sangre venosa de pacientes diagnosticados con Diabetes de tipo II como control negativo y en pacientes diabéticos con enfermedad renal crónica antes de iniciar el tratamiento de diálisis y luego durante el tratamiento. Se utilizó el test de t- Student para muestras independientes y emparejadas. Resultados: Se observó un aumento significativo de daño basal y oxidativo en el material genético de pacientes diabéticos con enfermedad renal crónica, comparados con los controles negativos (p< 0.005) y se observó, además, que el daño celular aumenta con el tratamiento de hemodiálisis (p<0.005). Conclusión: Los resultados obtenidos en esta investigación permiten concluir que el estrés oxidativo tiene un efecto genotóxico y que el nivel de daño genético es un buen bioindicador del avance de la enfermedad renal crónica y que la hemodiálisis induce a un aumento de daño a nivel del material genético, aumentando el riesgo de carcinogénesis.


Introduction: Processes such as mutagenesis, carcinogenesis and teratogenesis are the product of the interaction of agents of endogenous and exogenous origin that interact with the DNA molecule in a chronic way producing ruptures in the double helix, and in complete chromosomes resulting in genomic instability. The oxidative stress to which the cells are subjected when reactive oxygen species (ROS) and reactive nitrogen species (RNS) are formed, which may come from radicals produced as a result of diabetes or in initial stages of chronic kidney disease or in response to inflammatory processes in advanced stages of these pathologies, act as endogenous genotoxic agents. Objectives: This research aimed to determine the basal damage in the DNA molecule of hemodialyzed diabetic patients, through the Comet assay, as a bioindicator of genomic instability, during six months of treatment. Materials and methods: For this research, a prospective longitudinal cohort study was proposed to compare the different levels of genetic damage before and during the first six of hemodialysis treatment. Baseline damage was evaluated with the comet test or single cell electrophoresis, in venous blood samples from patients diagnosed with Type II Diabetes as a negative control and in diabetic patients with chronic kidney disease before starting dialysis treatment and then during treatment. Results: A significant increase in basal and oxidative damage was observed in the genetic material of diabetic patients with chronic kidney disease, compared to negative controls (p< 0.005) and it was also observed that cell damage increases with hemodialysis treatment (p<0.005). The t-Student test was used for independent and paired samples. Conclusion: The results obtained in this research allow us to conclude that oxidative stress has a genotoxic effect and that the level of genetic damage is a good bioindicator of the progression of chronic kidney disease and that hemodialysis induces an increase in damage at the level of the genetic material, increasing the risk of carcinogenesis.


Subject(s)
Renal Dialysis , Comet Assay , Dialysis , Research , DNA , Oxidative Stress
3.
Braz. J. Pharm. Sci. (Online) ; 58: e20575, 2022. tab, graf
Article in English | LILACS | ID: biblio-1420470

ABSTRACT

Abstract The composition and pharmacological properties of Lippia alba (Mill.) (L. alba) (Verbenaceae) flower and leaf essential oils (EO) were determined in this study. The major constituents in the flower EO were geranial (49.83%) and neral (32.75%), and in the leaf EO were geranial (38.06%), neral (31.02%), and limonene (18.03%). Flower EO inhibited thrombolysis induced by Bothrops moojeni (B. moojeni) and Lachesis muta muta (L. muta muta) venoms (0.05-1.2 µL mL-1). When tested against L. muta muta venom, the protective effect was smaller in both EO. The EOs prolonged the clotting time induced by L. muta muta venom and a procoagulant effect was observed on B. moojeni. In the comet assay, the flower EO presented anti-genotoxic action (damage frequency of only 11.6 - 34.9%) against the L. muta muta venom. The positive control (Doxorubicin) and the venom alone presented a damage frequency of 80.3% and 70.7%, respectively. The flower EO protected DNA from damage induced by L. muta muta venom. L. alba leaf and flower EOs presented anti-genotoxic action


Subject(s)
Biological Products/analysis , Oils, Volatile/analysis , Lippia/adverse effects , Plant Leaves/classification , Comet Assay/instrumentation , Flowers/classification , Elapid Venoms/pharmacology , Enzyme Inhibitors/administration & dosage , Hemostasis
4.
Article in English | LILACS, BBO | ID: biblio-1250451

ABSTRACT

ABSTRACT Objective: To evaluate genotoxicity of zinc oxide, P. A. calcium hydroxide, mineral trioxide aggregate and an iodoform paste using comet assay on human lymphocytes. Material and Methods: Two positive controls were used: methyl-methanesulfonate for the P.A. calcium hydroxide and mineral trioxide aggregate; and doxorubicin for the iodoform paste and zinc oxide. There were also two negative controls: distilled water for the P.A. calcium hydroxide and mineral trioxide aggregate; and DMSO for the iodoform paste and zinc oxide. Comets were identified using fluorescence microscopy and 100 of them were counted on each of the three slides analyzed per drug test. A damage index was established, taking into consideration the score pattern that had previously been determined from the size and intensity of the comet tail. Analysis of variance, followed by Tukey's test, was used to compare the means of the DNA damage indices. Results: The DNA damage index observed for mineral trioxide aggregate (7.08 to 8.58) and P.A. calcium hydroxide (6.50 to 8.33), which were similar to negative control index. On the other hand, damage index for zinc oxide (104.7 to 218.50) and iodoform paste (115.7 to 210.7) were similar to positive control index. Conclusion: Iodoform paste and zinc oxide showed genotoxicity at all concentrations used.


Subject(s)
Humans , Tooth, Deciduous , Zinc Oxide , Comet Assay , Genotoxicity , Mutagenicity Tests/instrumentation , Zinc Oxide , Brazil , Calcium Hydroxide , Analysis of Variance , Microscopy, Fluorescence
5.
Arq. bras. med. vet. zootec. (Online) ; 72(3): 853-861, May-June, 2020. ilus, tab, graf
Article in English | LILACS, VETINDEX | ID: biblio-1129489

ABSTRACT

The present study tested a comet assay that was modified for compatibility with Giemsa staining to assess the drug genotoxicity in the peripheral blood of rats. We analysed the peripheral blood of 16 female Wistar rats (N=8 rats/group) from a control group and from a group that was treated with an intraperitoneal injection of 50mg cyclophosphamide/kg. The comet assay was carried out with modifications of the blood volume and immersion time in the lysing solution and different combinations of electrophoresis conditions (running time, voltage and current), to Giemsa staining. The lysing time and electrophoresis conditions allowed for the expression of all classes of DNA damage during the electrophoresis run, and the comets were efficiently stained with Giemsa. The technique showed high reproducibility for the DNA classes. The results demonstrate that the modified comet assay with Giemsa staining can be standardized for routine laboratory procedures using a 20µL blood sample, 3h and 30min immersions in the lysing solution and electrophoresis runs with 23 to 25 V and 310 and 360mA of electrical current. The modified comet assay with Giemsa staining that was described in the present study was standardized to be applied in the laboratory routine.(AU)


O presente estudo testou um ensaio cometa modificado para a coloração de Giemsa para avaliar a genotoxicidade de fármacos no sangue periférico de ratos. Analisou-se o sangue periférico de 16 ratas Wistar (n=8 ratas/grupo) de um grupo controle e de um grupo que foi tratado com uma injeção intraperitoneal de 50mg/kg pv. de ciclofosfamida. O ensaio cometa foi realizado com modificações do volume sanguíneo e do tempo de imersão na solução de lise, bem como com diferentes combinações de condições de eletroforese (tempo de corrida, tensão e corrente), para coloração de Giemsa. O tempo de lise e as condições de eletroforese permitiram a expressão de todas as classes de danos no DNA durante a corrida de eletroforese, e os cometas foram eficientemente corados com Giemsa. A técnica mostrou alta reprodutibilidade para as classes de DNA. Os resultados demonstram que o ensaio cometa modificado com coloração de Giemsa foi padronizado para procedimentos laboratoriais de rotina usando-se uma amostra de sangue de 20µL, 3h30min de imersão na solução de lise e eletroforese com 23 a 25 V e 310 e 360mA. O ensaio cometa modificado com coloração de Giemsa descrito foi padronizado para ser aplicado na rotina laboratorial.(AU)


Subject(s)
Animals , Rats , Staining and Labeling/veterinary , Azure Stains/toxicity , Comet Assay/veterinary , Genotoxicity/analysis , Electrophoresis/veterinary , Mutagenicity Tests/veterinary
6.
Arq. bras. cardiol ; 114(2): 234-242, Feb. 2020. tab, graf
Article in English | LILACS | ID: biblio-1088869

ABSTRACT

Abstract Background: Chronic heart failure (CHF) is a complex syndrome which comprises structural and functional alterations in the heart in maintaining the adequate blood demand to all tissues. Few investigations sought to evaluate oxidative DNA damage in CHF. Objective: To quantify the DNA damage using the comet assay in left ventricle (LV), lungs, diaphragm, gastrocnemius and soleus in rats with CHF. Methods: Twelve male Wistar rats (300 to 330 g) were selected for the study: Sham (n = 6) and CHF (n = 6). The animals underwent myocardial infarction by the ligation of the left coronary artery. After six weeks, the animals were euthanized. It was performed a cell suspension of the tissues. The comet assay was performed to evaluate single and double strand breaks in DNA. Significance level (p) considered < 0.05. Results: The CHF group showed higher values of left ventricle end-diastolic pressure (LVEDP), pulmonary congestion, cardiac hypertrophy and lower values of maximal positive and negative derivatives of LV pressure, LV systolic pressure (p < 0.05). CHF group showed higher DNA damage (% tail DNA, tail moment and Olive tail moment) compared to Sham (p < 0.001). The tissue with the highest damage was the soleus, compared to LV and gastrocnemius in CHF group (p < 0.05). Conclusion: Our results indicates that the CHF affects all tissues, both centrally and peripherically, being more affected in skeletal muscle (soleus) and is positively correlated with LV dysfunction.


Resumo Fundamento: A insuficiência cardíaca crônica (ICC) é uma síndrome complexa que compreende alterações estruturais e funcionais no coração, mantendo demanda sanguínea adequada a todos os tecidos. Poucas investigações procuraram avaliar o dano oxidativo ao DNA na ICC. Objetivo: Quantificar o dano ao DNA utilizando o ensaio cometa no ventrículo esquerdo (VE), pulmões, diafragma, gastrocnêmio e sóleo em ratos com ICC. Métodos: Doze ratos Wistar machos (300 a 330 g) foram selecionados para o estudo: placebo (n = 6) e ICC (n = 6). Os animais foram submetidos a infarto do miocárdio através de ligadura da artéria coronária esquerda. Após seis semanas, os animais foram sacrificados. Foi realizada uma suspensão celular dos tecidos. O ensaio cometa foi realizado para avaliar as quebras de fita simples e dupla no DNA. Nível de significância (p) < 0,05. Resultados: O grupo ICC apresentou maiores valores de pressão diastólica final do ventrículo esquerdo (PDFVE), congestão pulmonar, hipertrofia cardíaca e menores valores de derivados máximos positivos e negativos da pressão do VE, pressão sistólica do VE (p < 0,05). O grupo ICC apresentou maior dano ao DNA (% de DNA da cauda, momento da cauda e momento da cauda de Olive) em comparação ao placebo (p < 0,001). O tecido com maior dano foi o sóleo, comparado ao VE e ao gastrocnêmio no grupo ICC (p < 0,05). Conclusão: Nossos resultados indicam que a ICC afeta todos os tecidos, de maneira central e periférica, sendo mais afetada no músculo esquelético (sóleo) e está positivamente correlacionada com a disfunção do VE.


Subject(s)
Animals , Male , DNA Damage/genetics , Heart Failure/genetics , Reference Values , Rats, Wistar , Oxidative Stress , Muscle, Skeletal/pathology , Comet Assay , Single-Cell Analysis , Heart Failure/pathology , Heart Ventricles/pathology , Hemodynamics , Liver/pathology , Lung/pathology , Myocardial Infarction/genetics , Myocardial Infarction/pathology
7.
São Paulo; s.n; s.n; 2020. 172 p. tab, graf.
Thesis in Portuguese | LILACS | ID: biblio-1292637

ABSTRACT

O câncer de pâncreas (PC) é uma das doenças mais devastadoras com o pior resultado de sobrevivência quando comparada com outros tipos de câncer. É apontado como o décimo câncer mais comum e a quarta causamortis por câncer, projetando-se para ser a segunda até 2030 nos Estados Unidos e na Alemanha. O PC constitui um conjunto heterogêneo de tumores, o adenocarcinoma ductal (PDAC) constitui o tipo mais frequente da neoplasia (80%). A prevenção, detecção precoce e tratamento enfrentam sérios problemas e é urgente a identificação de novos marcadores para diagnóstico, prognóstico e estratégias terapêuticas da doença. O objetivo desse trabalho foi realizar uma análise com alta-resolução do transcriptoma do PDAC) para identificar novos RNAs não codificadores, variantes de splicing e alterações transcricionais associados à malignidade. A metodologia empregada constituiu-se de gerar bibliotecas de RNA total a partir de 14 amostras cirúrgicas pareadas de tecido tumoral (PDAC) e tecido não-tumoral adjacente para sequenciamento NGS. A partir dos dados de RNAseq foi realizada a montagem do transcriptoma utilizando-se a referência do catálogo GENCODE para classificar os transcritos. Seguiram-se análises subsequentes de avaliação de características dos transcritos: estruturais, marcas regulatórias, potencial codificador, detecção em banco de dados independente (miTrascriptome, miT). Em segundo lugar foram realizadas análises de expressão diferencial, análise de sobrevida (utilizando banco de dados públicos) para seleção de transcritos potencialmente relevantes no PDAC. Foram geradas redes de co-expressão gênicas com enriquecimento de funções biológicas. Uma série de validações foram realizadas por RT-PCR de transcritos novos intergênicos e novas formas de splicing reconstruídas e selecionadas. RT-qPCR foi utilizada para validação da expressão aberrante de lncRNAs em PDAC, silenciamentos por siRNA e subsequentes ensaios de proliferação, migração e invasão. Foi avaliada in vivo o envolvimento com crescimento tumoral por ensaio xenográfico, e avaliação da implicação em funções biológicas mais específicas, como envolvimento em reparo de DNA por ensaio cometa alcalino e avaliação de enriquecimento em tumoresferas. A montagem reconstruiu 90.522 transcritos, dos quais 41.341 são anotados no GENCODE e 6.710 transcritos são novos não anotados no GENCODE (classificado como splicingvariant, intergenic RNA, antisense RNA). Desses foram validados 6 novos lincRNAs com expressão aumentada em PDAC, dois deles (TCONS00085964 e TCONS00036574) tem a expressão correlacionada com alterações na sobrevida. Foram validadas também novas formas de splicing de MMP14, CAPN8, LIF e OCT3 com expressão aumentada em PDAC. 7 lncRNAs, anotados no GENCODE, com expressão aumentada em PDAC, desses foram implicados com fenótipo tumoral de migração, invasão e proliferação: LINC01559; LINC01133, CCAT1 e UCA1. Desses LINC01559 e CCAT1 demonstraram regular a expressão de enzimas de O-glicosilação (GALNT3 e B3GNT3) envolvidas na manutenção de células tronco-tumorais em PDAC. O lncRNA UCA1 está envolvido com reparo de DNA e envolvido com a progressão tumoral invivo. Conclui-se que a abordagem por amostras pareadas a partir de RNAseq de bibliotecas de RNA total gerou um catálogo de transcritos mais completo no PDCA e revelou IncRNAs funcionalmente implicadas na doença como LINC01559 e UCA1, ampliando o conhecimento sobre os mecanismos moleculares que sustentam fenótipos malignos no câncer de pâncreas e revelando novos biomarcadores para prognóstico


Tese de DoutoradoDOIhttps://doi.org/10.11606/T.46.2019.tde-09032020-085211DocumentoTese de DoutoradoAutorPaixão, Vinicius Ferreira da (Catálogo USP)Nome completoVinicius Ferreira da PaixãoE-mailE-mailUnidade da USPInstituto de QuímicaÁrea do ConhecimentoBioquímicaData de Defesa2019-12-04ImprentaSão Paulo, 2019OrientadorReis, Eduardo Moraes (Catálogo USP) Banca examinadoraReis, Eduardo Moraes (Presidente) Hajj, Glaucia Noeli Maroso Malnic, Bettina Panepucci, Rodrigo Alexandre Título em portuguêsAnotação e caracterização de novos transcritos expressos no adenocarcinoma de pâncreas: RNAS não-codificadores longos associados a fenótipos tumorais e clínicos e formas alternativas de splicingPalavras-chave em portuguêsAlternative splicing lncRNA PDAC Transcriptoma UCA1 Xenotumor Resumo em portuguêsO câncer de pâncreas (PC) é uma das doenças mais devastadoras com o pior resultado de sobrevivência quando comparada com outros tipos de câncer. É apontado como o décimo câncer mais comum e a quarta causamortis por câncer, projetando-se para ser a segunda até 2030 nos Estados Unidos e na Alemanha. O PC constitui um conjunto heterogêneo de tumores, o adenocarcinoma ductal (PDAC) constitui o tipo mais frequente da neoplasia (80%). A prevenção, detecção precoce e tratamento enfrentam sérios problemas e é urgente a identificação de novos marcadores para diagnóstico, prognóstico e estratégias terapêuticas da doença. O objetivo desse trabalho foi realizar uma análise com alta-resolução do transcriptoma do PDAC) para identificar novos RNAs não codificadores, variantes de splicing e alterações transcricionais associados à malignidade. A metodologia empregada constituiu-se de gerar bibliotecas de RNA total a partir de 14 amostras cirúrgicas pareadas de tecido tumoral (PDAC) e tecido não-tumoral adjacente para sequenciamento NGS. A partir dos dados de RNAseq foi realizada a montagem do transcriptoma utilizando-se a referência do catálogo GENCODE para classificar os transcritos. Seguiram-se análises subsequentes de avaliação de características dos transcritos: estruturais, marcas regulatórias, potencial codificador, detecção em banco de dados independente (miTrascriptome, miT). Em segundo lugar foram realizadas análises de expressão diferencial, análise de sobrevida (utilizando banco de dados públicos) para seleção de transcritos potencialmente relevantes no PDAC. Foram geradas redes de co-expressão gênicas com enriquecimento de funções biológicas. Uma série de validações foram realizadas por RT-PCR de transcritos novos intergênicos e novas formas de splicing reconstruídas e selecionadas. RT-qPCR foi utilizada para validação da expressão aberrante de lncRNAs em PDAC, silenciamentos por siRNA e subsequentes ensaios de proliferação, migração e invasão. Foi avaliada in vivo o envolvimento com crescimento tumoral por ensaio xenográfico, e avaliação da implicação em funções biológicas mais específicas, como envolvimento em reparo de DNA por ensaio cometa alcalino e avaliação de enriquecimento em tumoresferas. A montagem reconstruiu 90.522 transcritos, dos quais 41.341 são anotados no GENCODE e 6.710 transcritos são novos não anotados no GENCODE (classificado como splicingvariant, intergenic RNA, antisense RNA). Desses foram validados 6 novos lincRNAs com expressão aumentada em PDAC, dois deles (TCONS00085964 e TCONS00036574) tem a expressão correlacionada com alterações na sobrevida. Foram validadas também novas formas de splicing de MMP14, CAPN8, LIF e OCT3 com expressão aumentada em PDAC. 7 lncRNAs, anotados no GENCODE, com expressão aumentada em PDAC, desses foram implicados com fenótipo tumoral de migração, invasão e proliferação: LINC01559; LINC01133, CCAT1 e UCA1. Desses LINC01559 e CCAT1 demonstraram regular a expressão de enzimas de O-glicosilação (GALNT3 e B3GNT3) envolvidas na manutenção de células tronco-tumorais em PDAC. O lncRNA UCA1 está envolvido com reparo de DNA e envolvido com a progressão tumoral invivo. Conclui-se que a abordagem por amostras pareadas a partir de RNAseq de bibliotecas de RNA total gerou um catálogo de transcritos mais completo no PDCA e revelou IncRNAs funcionalmente implicadas na doença como LINC01559 e UCA1, ampliando o conhecimento sobre os mecanismos moleculares que sustentam fenótipos malignos no câncer de pâncreas e revelando novos biomarcadores para prognóstico.Título em inglêsDetermination of relevant transcripts in ductal adenocarcinoma of pancreas: the transcriptional landscape of the long non-coding RNAs and protein-encoding RNAs revealed by the total RNAseq analysis of pancreatic adenocarcinomaPalavras-chave em inglêsAlternative splicing lncRNA PDAC Transcriptome UCA1 Xenotumor Resumo em inglêsPancreatic cancer (PC) is the deadliest malignancy, one of the most devastating diseases with the worst survival outcome compared to any cancer. It is touted as the tenth most common cancer and the fourth leading cause of cancer in the United States and Germany. It is projected to be the second leading cause of cancer death in a decade. PC is a heterogeneous set of tumors with high aggressiveness and mortality. Of these tumors, ductal adenocarcinoma (PDAC) is the most frequent type of cancer (80%). Prevention, early detection and treatment face serious problems, and the identification of new markers for diagnosis, prognosis and therapeutic strategies of the disease is urgent. The aim of this study was to perform a high-resolution analysis of pancreatic adenocarcinoma (PDAC) transcriptome to identify new noncoding RNAs, splicing variants, and transcriptional changes associated with malignancy in pancreatic ductal adenocarcinoma. The methodology employed consisted of generating total RNA libraries from 14 paired surgical samples of tumor tissue (PDAC) and adjacent non-tumor tissue for NGS sequencing. From the RNAseq data, the transcriptome assembly was performed using the GENCODE catalog reference to classify the transcripts. Subsequent analyzes of evaluation of transcript characteristics were followed: structural, regulatory markers, potential encoder, independent database detection (miTrascriptome, miT). Secondly, differential expression analysis, survival analysis (using public database) were performed to select potentially relevant transcripts in the PDAC. Genic co-expression networks with biological function enrichment were generated. A series of validations were performed by RT-PCR of new intergenic transcripts and new forms of reconstructed and selected splicing. RT-qPCR was used for validation of aberrant expression of lncRNAs in PDAC, siRNA silencing and subsequent proliferation, migration and invasion assays. Involvement with tumor growth was evaluated by in vivo xenograph assay. Biological function tests to determine an involvement in DNA repair was evaluated by alkaline comet assay and was performed an evaluation of tumorsphere expression enrichment. The assembly reconstructed a total of 90,522 transcripts, of which 41,341 are noted in GENCODE. 6,710 transcripts are new (splicing variant, intergenic RNA, antisense RNA) not noted in the GENCODE reference. Of these 6 new PDAC-enhanced lincRNAs were validated, two of them (TCONS00085964 and TCONS00036574) correlated with changes in survival. New forms of splicing of MMP14, CAPN8, LIF and OCT3 with increased expression in PDAC were also validated. 7 lncRNAs noted with increased expression in PDAC were implicated with tumor migration, invasion and proliferation phenotype: LINC01559; LINC01133, LINC01614; CCAT1; LINC02577; LINC00920 and UCA1. Of these LINC01559 has been shown to regulate the expression of O-glycosylation enzymes (GALNT3 and B3GNT3) involved in maintaining CSCs in PDAC. It has been identified that UCA1 is involved with DNA repair and involved with tumor progression in vivo. It is concluded that the approach of sampling RNAseq from total RNA libraries generated a more accurate transcript catalog of PDAC and revealed IncRNAs functionally implicated in the disease, such as LINC01559 and UCA1, expanding the knowledge about the molecular mechanisms that underlie malignant phenotypes in pancreatic cancer and revealing novel prognostic biomarke


Subject(s)
Pancreas , RNA , RNA, Antisense , Transcriptome , RNA, Long Noncoding , Referral and Consultation , Stem Cells , Comet Assay , Diagnosis , Enzymes
8.
Biomédica (Bogotá) ; 39(3): 464-477, jul.-set. 2019. tab, graf
Article in Spanish | LILACS | ID: biblio-1038807

ABSTRACT

Resumen Introducción. La exposición a solventes orgánicos y pinturas se ha asociado con efectos genotóxicos y mayor riesgo de neoplasias. Sin embargo, aún no se ha caracterizado bien el tipo de daño que esta exposición induce en el ADN humano, ni los mecanismos por los cuales se genera. Uno de los grupos con mayor exposición a dichos solventes y pinturas son los pintores de automóviles del sector informal que trabajan sin adecuadas prácticas de seguridad ocupacional. Objetivo. Determinar el daño oxidativo y por metilación del ADN de linfocitos de pintores de automóviles expuestos a solventes orgánicos y pinturas. Materiales y métodos. Se analizaron linfocitos aislados de sangre periférica de 62 pintores y 62 sujetos no expuestos mediante el ensayo cometa de gran eficiencia acoplado a las enzimas Fpg y AlkA. Las categorías de daño en el ADN evaluadas fueron el daño basal (sin enzimas), el daño oxidativo y el daño por metilación, y el parámetro de medición, el porcentaje de ADN en la cola. Resultados. El porcentaje de ADN en la cola fue mayor en el grupo expuesto con respecto al no expuesto (p<0,05). En el grupo expuesto, dicho porcentaje fue mayor en la categoría de daño oxidativo comparado con la del basal (16,50 Vs. 12,87; p<0,001), en tanto que en el daño por metilación no se encontraron diferencias significativas (14,00 Vs. 12,87; p>0,05). Conclusión. La exposición a solventes orgánicos y pinturas se asoció con el aumento de las lesiones oxidativas del ADN de los linfocitos de pintores de automóviles, tales como la producción de 8-oxo-2'-desoxiguanosina (8-oxodG) y otros productos formamidopirimidina, los cuales se consideran considerablemente mutagénicos.


Abstract Introduction: The exposure to organic solvents and paints has been associated with genotoxicity and a greater risk of neoplasms. However, the type of DNA damage induced in humans by the exposure to these compounds, which would help explain the mechanisms of their genotoxicity, is still not fully characterized. Due to inadequate practices of occupational safety, car painters in the informal sector are a highly exposed group to organic solvents and paints. Objective: To identify the oxidative and methylating damage in the DNA of lymphocytes of car painters exposed to organic solvents and paints. Materials and methods: Isolated peripheral blood lymphocytes from 62 painters and 62 unexposed subjects were analyzed by the modified high-throughput comet assay with the Fpg and AlkA enzymes. The categories used for the evaluation of the DNA damage were basal damage (without enzymes), oxidative and methylating damage. The measurement parameter used to establish the damage was the percentage of DNA in the tail. Results: The percentage of DNA in the tail was higher in the exposed group compared to the unexposed group (p<0.05). In the exposed group, this percentage was higher in the oxidative damage category than the baseline (16.50 vs. 12.87; p<0.001), whereas methylating damage did not show significant differences (14.00 vs. 12.87; p>0.05). Conclusion: In this study, exposure to organic solvents and paints was associated with an increase in oxidative lesions in the DNA of car painters' lymphocytes, such as the production of 8-oxodG and other formamidopyrimidine products which are considered highly mutagenic.


Subject(s)
Adult , Humans , Male , Middle Aged , Young Adult , Paint/toxicity , Solvents/toxicity , DNA Damage , Occupational Exposure/adverse effects , Oxidative Stress , DNA Methylation , Automobiles , DNA/drug effects , Lymphocytes/drug effects , Case-Control Studies , Cell Survival , Cross-Sectional Studies , Comet Assay , Mutagens/toxicity
9.
Braz. j. oral sci ; 18: e191430, jan.-dez. 2019. ilus
Article in English | LILACS, BBO | ID: biblio-1095234

ABSTRACT

Aim: DNA damage associated with Oral Squamous Cell Carcinoma (OSCC) and potentially malignant disorders (PMDs) is produced due to carcinogenic agents or increased oxidative stress. Comet assay can assist in early detection and evaluation of the amount of DNA damage; lymphocytesare the most commonly used cells for performing comet assay. Utilisation of buccal epithelial cells in comet assay can be a minimally invasive and rapid method. The present study compared the efficacy of comet assay in assessing DNA damage in buccal cells over peripheral blood leucocytes (PBLs) in oral potentially malignant and malignant disorders. Methods: The study included fifty five patients each of Leukoplakia, Oral Submucous Fibrosis (OSMF) and OSCC along with fifty five healthy individuals as control. Buccal epithelial cells were collected from all the selected subjects. DNA damage was evaluated bymeasuring the mean tail length (µm). Results: A significantly increased mean tail length (µm) and higher DNA damage were found in OSCC (26.1096 + 1.84355) and there was a progressive stepwise increase in mean tail length from control(8.4982 + 0.93307) to PMD [leukoplakia (14.6105 + 0.71857); OSMF (12.5009 + 1.12694)] to OSCC.The mean tail length in different habit groups was greater than controls, though no significant difference was noted between habit groups. The mean tail length of buccal cells was significantly greater than the mean tail length of PBLs in all study groups and controls. Conclusion: Hence, use of comet assay on buccal epithelial cells can prove to be beneficiary for evaluation of DNA damage


Subject(s)
Humans , Male , Female , DNA Damage , Mouth Neoplasms , Comet Assay , Epithelial Cells , Leukocytes
10.
Journal of Nutrition and Health ; : 413-421, 2019.
Article in Korean | WPRIM | ID: wpr-766003

ABSTRACT

PURPOSE: Astragalus membranaceus (AM) is an important traditional medicinal herb. Pharmacological research has indicated that AM has various physiological activities such as antioxidant, anti-inflammatory, immunoregulatory, anticancer, hypolipidemic, antihyperglycemic, and hepatoprotective activities. The bioactive substances responsible for the physiological activities in AM, including many antioxidant substances, change during the roasting process. This study investigated and compared the changes in the antioxidant constituents of AM caused by roasting. METHODS: DPPH (1,1-diphenyl-2-picryl hydrazyl) and ABTS⁺ (2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) radical scavenging activities and their total phenolic content (TPC) were measured. High-performance liquid chromatography (HPLC) analysis was performed to confirm any changes in the isoflavonoids of roasted AM (R-AM),. The cell viability of UVB-induced HDF (Human dermal fibroblast) cells treated with AM and R-AM extracts was investigated. The comet assay was used to examine the inhibitory effects of R-AM extracts on DNA damage caused by oxidative stress. RESULTS: The DPPH and ABTS⁺ radical scavenging activities were 564.6±20.9 and 108.2±3.1 (IC₅₀ value) respectively, from the 2R-AM. The total phenol content was 47.80±1.40 mg GAE/g from the 1R-AM. The values of calycosin and formononetin, which are the known isoflavonoid constituents of AM, were 778.58±2.72 and 726.80±3.45 µg/g respectively, from the 2R-AM. Treatment of the HDF cells with R-AM (50 ~ 200 µg/mL) did not affect the cell viability. Furthermore, the R-AM extracts effectively protected against UVB-induced DNA damage. CONCLUSION: The findings of this study indicate that R-AM increases its isoflavonoid constituents and protects against UVB-induced DNA damage in HDF cells.


Subject(s)
Astragalus propinquus , Cell Survival , Chromatography, Liquid , Comet Assay , DNA Damage , Oxidative Stress , Phenol , Plants, Medicinal
11.
Cancer Research and Treatment ; : 1167-1179, 2019.
Article in English | WPRIM | ID: wpr-763163

ABSTRACT

PURPOSE: The DNA damage response (DDR) is a multi-complex network of signaling pathways involved in DNA damage repair, cell cycle checkpoints, and apoptosis. In the case of biliary tract cancer (BTC), the strategy of DDR targeting has not been evaluated, even though many patients have DNA repair pathway alterations. The purpose of this study was to test the DDR-targeting strategy in BTC using an ataxia-telangiectasia and Rad3-related (ATR) inhibitor. MATERIALS AND METHODS: A total of nine human BTC cell lines were used for evaluating anti-tumor effect of AZD6738 (ATR inhibitor) alone or combination with cytotoxic chemotherapeutic agents through MTT assay, colony-forming assays, cell cycle analyses, and comet assays. We established SNU478-mouse model for in vivo experiments to confirm our findings. RESULTS: Among nine human BTC cell lines, SNU478 and SNU869 were the most sensitive to AZD6738, and showed low expression of both ataxia-telangiectasia mutated (ATM) and p53. AZD6738 blocked p-Chk1 and p-glycoprotein and increased γH2AX, a marker of DNA damage, in sensitive cells. AZD6738 significantly increased apoptosis, G2/M arrest and p21, and decreased CDC2. Combinations of AZD6738 and cytotoxic chemotherapeutic agents exerted synergistic effects in colony-forming assays, cell cycle analyses, and comet assays. In our mouse models, AZD6738 monotherapy decreased tumor growth and the combination with cisplatin showed more potent effects on growth inhibition, decreased Ki-67, and increased terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling than monotherapy with each drug. CONCLUSION: In BTC, DDR targeting strategy using ATR inhibitor demonstrated promising antitumor activity alone or in combination with cytotoxic chemotherapeutic agents. This supports further clinical development of DDR targeting strategy in BTC.


Subject(s)
Animals , Humans , Mice , Apoptosis , Ataxia Telangiectasia , Biliary Tract Neoplasms , Biliary Tract , Cell Cycle , Cell Cycle Checkpoints , Cell Line , Cisplatin , Comet Assay , DNA Damage , DNA Repair , DNA , ATP Binding Cassette Transporter, Subfamily B, Member 1
12.
Chinese Journal of Applied Physiology ; (6): 97-100, 2019.
Article in Chinese | WPRIM | ID: wpr-776552

ABSTRACT

OBJECTIVE@#To clarify the genotoxicity induced by acute exposure of ozone with different concentrations on pulmonary cells in rats.@*METHODS@#Thirty-six Wistar rats were randomly divided into control group (filtered air exposure) and ozone exposure group (0.12 ppm, 0.5 ppm, 1.0 ppm, 2.0 ppm, 4.0 ppm) with 6 in each group. After rats were exposed to different concentrations of ozone for 4 h, lung tissues were taken and single cells were isolated. Then, 8-hydroxydeoxyguanosine (8-OHdG) was quantitatively detected by enzyme-linked immunosorbent assay. Comet assay, micronucleus test and DNA- protein cross-linking assay were used to analyze DNA and chromosome damages.@*RESULTS@#Compared with the control group, the content of 8-OHdG in lung tissue was increased significantly from the ozone exposure concentration of 0.12 ppm, reaching the highest value at 0.5 ppm. With the increase of ozone exposure concentration, the tail rate of comets was increased gradually, and there was a significant dose-effect relationship. The cross-linking rate of DNA- protein was increased first and then was decreased with a maximum value at 2.0 ppm group. Although the micronucleus rate of lung cells showed an upward trend, there was no significant difference compared with the control group.@*CONCLUSION@#Acute exposure of ozone at low concentrations (0.12 ppm) could lead to DNA damage in the pulmonary cells of rats, while no significant chromosome damage was found even in the group with ozone concentration reached to 4 ppm.


Subject(s)
Animals , Rats , Comet Assay , DNA Damage , Lung , Cell Biology , Pathology , Micronucleus Tests , Ozone , Random Allocation , Rats, Wistar
13.
Rev. peru. med. exp. salud publica ; 35(3): 471-475, jul.-sep. 2018. tab, graf
Article in Spanish | LILACS | ID: biblio-978912

ABSTRACT

RESUMEN El objetivo del estudio fue evaluar el efecto de cloroquina (CQ) en linfocitos humanos a través del ensayo cometa. Los linfocitos fueron aislados de muestras de sangre periférica obtenidas de tres donantes sanos, no fumadores de 24 a 30 años. Los linfocitos aislados fueron expuestos durante una hora a diversos tratamientos: peróxido de hidrógeno 2,5 % (control positivo), buffer fosfato salino 1X (control negativo) y cloroquina a concentraciones de 0 µg/ml, 0,25 µg/ml; 5 µg/ml y 300 µg/ml. Se registró los promedios del porcentaje de ADN en la cola del cometa, momento de la cola y momento de la cola de Olive, encontrándose diferencias significativas entre las diversas concentraciones de CQ (p<0,01). Asimismo, la magnitud de daño del ADN se incrementó en función de la concentración de CQ. Se demostró el efecto genotóxico de CQ en linfocitos humanos expuestos in vitro.


ABSTRACT The objective of the study was to evaluate the effect of chloroquine (CQ) on human lymphocytes through the Comet Assay. Lymphocytes were isolated from peripheral blood samples obtained from three healthy, non-smoking donors aged 24 to 30 years. The isolated lymphocytes were exposed for one hour to various treatments: hydrogen peroxide 2.5% (positive control), saline buffer phosphate 1X (negative control) and chloroquine at concentrations of 0 µg/ml, 0.25 µg/ml; 5 µg/ml and 300 µg/ml. The averages of the percentage of DNA in the comet tail, moment of tail and moment of Olive's tail were recorded, with significant differences between the different concentrations of CQ (p<0.01). Also, the magnitude of DNA damage was increased as a function of the CQ concentration. The genotoxic effect of CQ was demonstrated in human lymphocytes exposed in vitro.


Subject(s)
Humans , DNA Damage , Lymphocytes , Chloroquine/adverse effects , Cells, Cultured , Cross-Sectional Studies , Comet Assay
14.
Bol. latinoam. Caribe plantas med. aromát ; 17(1): 68-83, ene. 2018. ilus, tab
Article in Spanish | LILACS | ID: biblio-915131

ABSTRACT

Los aceites esenciales (AEs), pertenecientes al género Lippia, son candidatos interesantes de formulaciones tópicas en el tratamiento de la leishmaniasis cutánea (LC). El objetivo de este trabajo fue determinar el perfil toxicológico y la actividad anti-Leishmania de AEs obtenidos de plantas colombianas del género Lippia. Ratones BALB/c fueron tratados tópica u oralmente con AEs obtenidos de L. alba quimiotipo citral (AE1) y de L. origanoides quimiotipos timol (AE2), carvacrol (AE3) y felandreno (AE4). El efecto del tratamiento en la irritación de la piel, la toxicidad aguda oral, la genotoxicidad (prueba cometa y micronúcleos), los cambios en la función hepática y renal, la inducción de reacción de hipersensibilidad de contacto y en la actividad contra L. (V) panamensis y L. (V.) braziliensis fueron determinados. Todos los AEs presentaron un perfil toxicológico similar a los parámetros normales, exceptuando los aceites AE2 y AE3 los cuales fueron irritantes y presentaron algunos signos de toxicidad aguda oral al ser utilizados en altas concentraciones (concentraciones bajas no fueron tóxicas). El AE2 mostró actividad antiparasitaria en las formas parasitarias evaluadas. Concentraciones bajas de los AEs podrían utilizarse de forma segura como componentes de formulaciones farmacológicas en LC.


Essential oils (EOs) belonging to the genus Lippia are interesting candidates in pharmaceutical systems for cutaneous leishmaniasis (CL). The aim of this work was to determine both toxicological and antileishmanial activities of EOs obtained from different species of Lippia, a widely distributed Colombian plants. BALB/c mice were treated topically or orally with EOs obtained from L. alba citral chemotype (EO1) and L. origanoides thymol (EO2), carvacrol (EO3) and phellandrene (EO4) chemotypes. The skin irritation, oral acute toxicity, genotoxicity (comet assay and micronucleus test), liver and renal adverse effects, All the EOs showed a toxicological profile similar to the normal parameters, except for oils EO2 and EO3 which were irritant and showed some signs of acute oral toxicity at high concentrations (low concentration were safe). The EO2 showed antiparasitic activity. Low concentrations of the EO could be used safely as components of pharmacological formulations in CL.


Subject(s)
Animals , Female , Mice , Oils, Volatile/pharmacology , Leishmaniasis, Cutaneous/drug therapy , Lippia/chemistry , Leishmania/drug effects , Antiprotozoal Agents/pharmacology , Oils, Volatile/adverse effects , Colombia , Comet Assay , Dermatitis, Contact/etiology , Genotoxicity , Mice, Inbred BALB C , Antiprotozoal Agents/adverse effects
15.
Clin. biomed. res ; 38(1): 50-57, 2018.
Article in English | LILACS | ID: biblio-994866

ABSTRACT

Introduction: Homocysteine (Hcy) tissue accumulation occurs in a metabolic disease characterized biochemically by cystathionine ß-synthase (CBS) deficiency and clinically by mental retardation, vascular problems, and skeletal abnormalities. Previous studies indicate the occurrence of DNA damage secondary to hyperhomocysteinemia and it was observed that DNA damage occurs in leukocytes from CBS-deficient patients. This study aimed to investigate whether an oxidative mechanism could be involved in DNA damage previously found and investigated the in vitro effect of N-acety-L-cysteine (NAC) on DNA damage caused by high Hcy levels. Methods: We evaluated a biomarker of oxidative DNA damage in the urine of CBS­deficient patients, as well as the in vitro effect of NAC on DNA damage caused by high levels of Hcy. Moreover, a biomarker of lipid oxidative damage was also measured in urine of CBS deficient patients. Results: There was an increase in parameters of DNA (8-oxo-7,8-dihydro-2'- deoxyguanosine) and lipid (15-F2t-isoprostanes levels) oxidative damage in CBS-deficient patients when compared to controls. In addition, a significant positive correlation was found between 15-F2t-isoprostanes levels and total Hcy concentrations. Besides, an in vitro protective effect of NAC at concentrations of 1 and 5 mM was observed on DNA damage caused by Hcy 50 µM and 200 µM. Additionally, we showed a decrease in sulfhydryl content in plasma from CBS-deficient patients when compared to controls. Discussion: These results demonstrated that DNA damage occurs by an oxidative mechanism in CBS deficiency together with lipid oxidative damage, highlighting the NAC beneficial action upon DNA oxidative process, contributing with a new treatment perspective of the patients affected by classic homocystinuria.


Subject(s)
Humans , Female , Child , Adolescent , Adult , Young Adult , Acetylcysteine/pharmacology , DNA Damage , Oxidative Stress , Cystathionine/metabolism , Deoxyguanosine/urine , Homocystinuria/genetics , Antioxidants/pharmacology , Biomarkers/urine , Case-Control Studies , Creatinine/urine , Comet Assay , Cystathionine/biosynthesis , Cystathionine/blood , Isoprostanes/analysis , Deoxyguanosine/analogs & derivatives , Homocysteine/blood , Homocystinuria/blood
16.
An. acad. bras. ciênc ; 89(4): 2657-2662, Oct.-Dec. 2017. tab
Article in English | LILACS | ID: biblio-886836

ABSTRACT

ABSTRACT The purpose of this study was to determine the effects of the high consumption of sucrose on the levels of DNA damage in blood, hippocampus and bone marrow of rats. Male Wistar rats were treated for 4 months with sucrose (10% for 60 initial days and 34% for the following 60 days) in drinking water, and then, glycemia and glycated hemoglobin (A1C) were measured. Levels of DNA damage in blood and hippocampus were evaluated by the comet assay. The micronucleus test was used to evaluate chromosomal damages in the bone marrow. The sucrose treatment significantly increased (p<0.01) the serum glucose levels (~20%) and A1C (~60%). The level of primary DNA damage was significantly increased (p<0.05) in hippocampal cells (~60%) but not in peripheral blood leukocytes (p>0.05). Additionally, it was observed a significative increase (p<0.05) in the markers of chromosomal breaks/losses in bone marrow, as indicated by the micronucleus test. This is the first study that evaluated DNA damage induced by high sucrose concentration in the hippocampus and bone marrow of rats. Sucrose-induced DNA damage was observed in both tissues. However, the mechanism of sucrose toxicity on DNA remains unknown.


Subject(s)
Animals , Male , Rats , Bone Marrow/drug effects , DNA Damage , Hippocampus/drug effects , Bone Marrow/pathology , Micronucleus Tests , Rats, Wistar , Dietary Sucrose/adverse effects , Comet Assay , Diabetes Mellitus, Type 2/complications , Disease Models, Animal , Hippocampus/pathology
17.
Biomédica (Bogotá) ; 37(3): 378-389, jul.-set. 2017. tab, graf
Article in Spanish | LILACS | ID: biblio-888478

ABSTRACT

Resumen Introducción. Dada la resistencia de Plasmodium a los medicamentos antipalúdicos, es necesario encontrar nuevas alternativas terapéuticas para su tratamiento y control. Con base en el saber indígena colombiano, se recopilaron extractos de plantas del Vaupés medio con potencial efecto antipalúdico. Objetivo. Evaluar el efecto mutagénico y genotóxico, y la expresión de los genes Rad51C, Xiap, P53 yNrf2, inducidos por cuatro extractos etanólicos con actividad anti-Plasmodium(R001, T002, T015 y T028). Materiales y métodos. Se evaluó el potencial mutagénico de cuatro extractos etanólicos con efecto antiplasmódico utilizando el test de Ames y el efecto genotóxico, con un ensayo del cometa; asimismo, se analizó la expresión de los genes Rad51C, Xiap, P53 y Nrf2 en células HepG2. Resultados. Los extractos no fueron mutágenos en la cepa TA98 de Salmonella typhimurium en presencia y ausencia de actividad metabólica de la fracción S9. En la cepa TA100, los extractos R001, T015 y T028 se comportaron como mutágenos débiles en presencia de S9, con índices mutagénicos de 1,58; 1,38; 1,53 y 1,61, respectivamente; T015 tuvo el mismo comportamiento en ausencia de S9, con un índice mutagénico de 1,36. En el ensayo del cometa, todos los extractos provocaron daño de categorías 1 o 2, con colas de cometas entre 36,7 y 51,48 µm de longitud; sin embargo, el índice dedaño genético sugirió que los tratamientos afectaron la mayoría de las células. En los genes en estudio, los extractos R001 y T028 indujeron una sobreexpresiónde 1,84 a 3,99 frente a las células sin tratar de los genes Xiap y P53. Conclusiones. Los resultados evidenciaron que el extracto T002 fue el más seguro, ya que presentó actividad anti-Plasmodium, no fue citotóxico en las células HepG2, no fue mutágeno, causó daño de categoría 1 en el ADN y no modificó la expresión de los genes evaluados.


Abstracts Introduction: Due to Plasmodium resistance to antimalarial drugs, it is important to find new therapeutic alternatives for malaria treatment and control. Based on the knowledge of Colombian indigenous communities, we collected extracts of plants with potential antimalarial effects from the middle Vaupés region. Objective: To evaluate the mutagenic and genotoxic effects, as well as the gene expression of Rad51C, Xiap, P53 and Nrf2 induced by four ethanolic extracts with antimalarial activity (R001, T002, T015 and T028). Materials and methods: We evaluated four ethanolic extracts with antimalarial activity using the Ames test to assess mutagenicity, and the comet assay on HepG2 cells to determine the genotoxicicity. We also evaluated the expression of Rad51C, Xiap, P53 and Nrf2 from HepG2 cells stimulated with the four extracts. Results: None of the four extracts was mutagenic in Salmonella typhimurium TA98 strain in the presence and absence of S9 metabolic activity. Extracts R001, T015 and T028 were weakly mutagenic on the TA100 strain in the presence of S9, with mutagenic indexes (MI) of 1.58, 1.53 and 1.61, respectively. The T015 strain showed the same behavior without S9 with an MI of 1.36. The results of the comet assay showed that the four extracts produced category 1 or 2 damage, with comets between 36.7 and 51.48 µm in length. However, the genetic damage index suggested that most of the cells were affected by the treatments. Regarding gene expression, extracts R001 and T028 induced an overexpression of genes Xiap and P53 with an 1.84 to 3.99 fold-change compared with untreated cells. Conclusions: These results revealed that the T002 extract was the safest as it had antimalarial activity and was not cytotoxic on HepG2 cells. Moreover, it was not mutagenic and it only produced category 1 damage on the DNA. Also, the extract did not induce a change in the expression of the tested genes.


Subject(s)
Humans , Plants, Medicinal/chemistry , Plant Extracts/pharmacology , Gene Expression Regulation/drug effects , Tumor Suppressor Protein p53/biosynthesis , DNA-Binding Proteins/biosynthesis , X-Linked Inhibitor of Apoptosis Protein/biosynthesis , NF-E2-Related Factor 2/biosynthesis , Antimalarials/pharmacology , Plasmodium falciparum/drug effects , Salmonella typhimurium/drug effects , Salmonella typhimurium/genetics , Solvents , Plant Extracts/isolation & purification , Tumor Suppressor Protein p53/genetics , Colombia , Comet Assay , Ethanol , DNA-Binding Proteins/genetics , Drug Evaluation, Preclinical , X-Linked Inhibitor of Apoptosis Protein/genetics , NF-E2-Related Factor 2/genetics , Hep G2 Cells , Activation, Metabolic , Genes, Bacterial/drug effects , Mutagenicity Tests , Antimalarials/isolation & purification
18.
Acta cir. bras ; 32(5): 388-395, May 2017. tab, graf
Article in English | LILACS | ID: biblio-837711

ABSTRACT

Abstract Purpose: To evaluate DNA damage levels in pregnant rats undergoing a treadmill exercise program. Methods: Wistar Kyoto rats were allocated into two groups (n= 5 animals/group): non-exercise and exercise. The pregnant rats were underwent an exercise protocol on a treadmill throughout pregnancy. Exercise intensity was set at 50% of maximal capacity during maximal exercise testing performed before mating. Body weight, blood pressure and glucose levels, and triglyceride concentration were measured during pregnancy. At day 10 post-natal, the animals were euthanized and maternal blood samples were collected for DNA damage. Results: Blood pressure and glucose levels and biochemical measurements showed no significant differences. Increased DNA damage levels were found in exercise group compared to those of non-exercise group (p<0.05). Conclusion: The exercise intensity protocol used in the study might have been exhaustive leading to maternal increased DNA damage levels, demonstrating the relevance of an adequate protocol of physical exercise.


Subject(s)
Animals , Female , DNA Damage/physiology , Exercise Test/adverse effects , Physical Conditioning, Animal , Rats, Inbred WKY , Blood Glucose/analysis , Blood Pressure/physiology , Body Weight/physiology , Pregnancy , Random Allocation , Comet Assay/methods , Models, Animal , Exercise Test/standards , Fetal Viability/physiology , Animals, Newborn/physiology
19.
Acta toxicol. argent ; 25(1): 1-11, mayo 2017. ilus, tab
Article in Spanish | LILACS | ID: biblio-886578

ABSTRACT

Los contaminantes del aire han sido y siguen siendo, los principales factores que contribuyen a las enfermedades crónicas como el asma y enfermedades cardiovasculares. La contaminación del aire por material particulado (PM) es un problema mundial y en los últimos años, el PM se ha convertido en un tema importante de investigación ya que tiene un impacto negativo significativo en la salud humana; el PM es generado por las actividades industriales y tubos de escape de vehículos de motor. Sin embargo, diversos componentes nocivos del PM, como los hidrocarburos aromáticos policiclicos (HAP) en general, son sos­pechosos de ser carcinogénicos. Este trabajo tiene como objetivo identificar los HAP presentes en el PM2.5 del aire de Cúcuta, extraídos por primera vez, mediante el sistema diclorometano-etanol-tolueno e investigar la importancia del fraccionamiento de la materia organica del PM2.5 para detectar los HAP presentes en las fracciones del PM2.5. La identificación de los HAP considerados como contaminantes prioritarios y reconocidos por su afectación a la salud de la población se realizó, mediante cromatografía de gases con detector FID. Los efectos genotoxicos de la materia orgánica del PM2.5 extraída con una mezcla de DCM-etanol-tolueno fueron evaluados mediante el ensayo Cometa.


Air pollutants have been and still are the main factors that contribute to chronic diseases such as asthma and cardio­vascular disease. Air pollution by particulate matter (PM) is a global problem and in recent years, the PM has become an important research topic since it has a significant negative impact on human health; the PM is generated by industrial activities and exhaust pipes of motor vehicles. However, various harmful components of PM such as polycyclic aromatic hydrocarbons (PAHs) in gen­eral, are suspected of being carcinogenic. This work aims to identify the PAHs present in the PM 2.5 air Cúcuta, first extracted by the dichloromethane-ethanol-toluene system and investigate the importance of organic matter fractionation of PM 2.5 to detect PAHs present in the fractions of PM 2.5. The identification of PAHs considered as priority pollutants and recognized for their effects on health of the population was performed by gas chromatography with FID detector. The genotoxic effects of PM2.5 organic mat­ter, extracted with a mixture of DCM-ethanol-toluene, was evaluated by the Comet assay.


Subject(s)
Humans , Carcinogens, Environmental , Genotoxicity , Polycyclic Aromatic Hydrocarbons/toxicity , Chemical Fractionation/methods , Colombia , Comet Assay/methods , Environmental Pollution
20.
Pesqui. vet. bras ; 37(3): 257-260, Mar. 2017. graf
Article in English | LILACS, VETINDEX | ID: biblio-842063

ABSTRACT

Fipronil is a pesticide widely used for controlling fleas and ticks in domestic animals, and its short-term exposure can lead to serious effects on animals. However, the possible genotoxic effect of this compound has not been investigated in target animals. Based on the hypothesis that fipronil can induce genotoxicity, this study evaluated the effect of fipronil on DNA damage in peripheral blood cells. For that purpose, ten dogs of both sexes were used in the study. The product (6.7mg/kg) was applied on the dorsal neck region of each animal. Peripheral blood samples were collected immediately prior to application of the product, and at 3, 8 and 24 hours after the application. Samples were processed for comet assay. No statistically significant differences were found among the four time points. The current study suggests for the first time that a single exposure to this pesticide does not induce systemic genotoxic effect in dogs.(AU)


O fipronil é um inseticida/herbicida amplamente utilizado para controle de pulgas e carrapatos em animais domésticos. Sua exposição a curto prazo tem acarretado efeitos deletérios em animais. Entretanto, o possível efeito genotóxico deste composto ainda não foi investigado em animais alvo. Baseando-se na hipótese de que o fipronil pode induzir genotoxicidade, o presente estudo avaliou o efeito deletério do fipronil no material genético de células de sangue periférico. Para isso, dez cães sadios, de ambos os sexos, foram utilizados neste estudo. O produto (6,7mg/kg) foi aplicado na região dorsal do pescoço de cada animal. As amostras de sangue foram coletadas imediatamente antes da aplicação do produto (controle) e após três, oito e 24 horas da aplicação. As amostras foram imediatamente processadas para condução do teste do cometa, a fim de se avaliar os danos basais no DNA. Não houve diferença significativa entre os quatro momentos de coleta em relação aos danos no material genético. O estudo sugere, pela primeira vez, que uma exposição única a este pesticida não induz efeito genotóxico sistêmico em cães.(AU)


Subject(s)
Animals , Dogs , Genotoxicity/analysis , Pesticides/analysis , Comet Assay/veterinary , DNA Damage
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