Análise da Expressão de APE1 após Estresse Oxidativo Induzido em Células Proficientes e Deficientes na Via de Reparo por Excisão de Nucleotídeos

Riboflavin is a vitamin very important in aerobic organisms, as a precursor of many coenzymes involved in the electron transporter chain. However, after photosensitization of riboflavin with UV or visible light, it generates reactive oxygen species (ROS), which can oxidize the DNA. The repair of oxidative lesions on DNA occurs through the base excision repair pathway (BER), where APE1 endonuclease plays a central role. On the other hand, the nucleotide excision repair pathway (NER) repairs helix-distorting lesions. Recently, it was described the participation of NERproteins in the repair of oxidative damage and in stimulation of repair function fromAPE1. The aim of this research was to evaluate the cytotoxic effects of photosensitized riboflavin (RF*) in cells proficient and deficient in NER, correlating with APE1 expression. For this propose, the cells were treated with RF* and it was performed the cell viability assay, extraction of whole proteins, cells fractionation, immunoblotting, indirect immunofluorescence and analysis of polymorphisms of BER gens. The results evidenced that cells deficient in XPA and CSB proteins were more sensitive to RF*. However, XPC-deficient cells presented similar resistance to MRC5- SV cells, which is proficient in NER. These results indicate that XPA and CSB proteins have an important role on repair of oxidative lesions induced by RF*. Additionally, it was evidenced that single nucleotide polymorphisms (SNPs) in BER enzymes may influence in sensitivity of NER-deficient cell lines. Concerning the APE1 expression, the results showed that expression of this protein after treatment with RF* only changed in XPC-deficient cells. Though, it was observed that APE1 is recruited and is bound to chromatin in MRC5-SV and XPA cells after treatment with RF*. The results also showed the induction of DNA damage after treatment with RF*, through the analysis of-H2AX, since the treatment promoted an increase of endogenous levels of this phosphorylated protein, which acts signaling double strand-break on DNA. On the other hand, in XPC-deficient cells, regardless of resistance of RF*, the endogenous levels of APE1 are extremely reduced when compared with other cell lines and APE1 is not bound to chromatin after treatment with RF*. These results conclude that RF* was able to induce cell death in NERdeficient cells, where XPA and CSB cells were more sensitive when compared with MRC5-SV and XPC-deficient cells. This last result is potentially very interesting, since XPC-deficient cell line presents low levels of APE1. Additionally, the results evidenced that APE1 protein can be involved in the repair of oxidative damage induced by RF*, because APE1 is recruited and bound strongly to chromatin after treatment.

Dermatose responsiva à biotina em cão

Os transtornos da pele e dos pelos são parte importante na prática clínica de pequenos animais. Numerosos fatores nutricionais afetam a homeostase, a qualidade e o aspecto da pelagem. As vitaminas do complexo B incluem compostos hidrossolúveis necessários como coenzimas em diversas funções celulares envolvidas no metabolismo energético e na síntese tecidual. A biotina, em especial, é necessária nas reações de carboxilação, participando da síntese de ácidos graxos, aminoácidos e purinas pelo tecido epitelial. Uma cadela com quadro de cistite recorrente e tumor venéreo transmissível foi tratada com antibioticoterapia prolongada e quimioterapia. Após alguns meses de tratamento, foram observadas lesões no plano nasal e nos coxins plantar e palmar, caracterizadas por hiperceratose, espessamento, fissuras, sangramento e inflamação. O paciente recebeu suplementação de 15mg de biotina por via oral (equivalente a 1,4mg kg-1 de peso corporal), uma vez por dia, durante 60 dias, havendo importante regressão das lesões. Sugere-se que, sob antibioticoterapia e doença, a síntese intestinal de biotina possa não ter sido suficiente, sendo necessária sua suplementação.

Efeito protetor da coenzima Q10 sobre os danos oxidativos da L-Tiroxina no músculo sóleo de ratos: Fabiana Barreiro de Freitas Silva. -

Pós-graduação em Ciência Animal - FMVA

Efeitos do cobre no metabolismo energético do bivalve marinho Mesodesma mactroides

Apesar de ser um micronutriente essencial aos organismos, o cobre (Cu) é tóxico quando presente em elevadas concentrações na água. O mecanismo pelo qual este metal exerce sua toxicidade em invertebrados marinhos ainda não está bem estabelecido. Dentre os diversos efeitos relatados, observa-se uma redução do consumo de oxigênio corporal e tecidual no marisco Mesodesma mactroides exposto (96 h) ao Cu (150 µg L-1 ) em água do mar (salinidade 30). Portanto, o objetivo do presente estudo foi avaliar os efeitos desta exposição ao Cu no metabolismo energético em teciduais do marisco M. mactroides. Os conteúdos de ATP e coenzimas (NAD+ e NADH) nas brânquias, glândula digestiva e músculo pedal não foram alterados pela exposição ao Cu, indicando que estes tecidos mantiveram suas capacidades de produção aeróbica de energia. Porém, foi observada uma redução no conteúdo hemolinfático de ATP. Quanto ao conteúdo de proteínas, houve um aumento na glândula digestiva, que pode estar associado à maior oxidação de proteínas já relatada para esse tecido após exposição ao Cu. Os conteúdos de lipídios, glicogênio e glicose permaneceram inalterados em todos os tecidos analisados, exceto no músculo pedal, onde foi observada uma redução no conteúdo de glicose. Por isso, os conteúdos de piruvato e lactato também foram analisados no músculo pedal e na hemolinfa. Em ambos tecidos, foi observado um aumento do conteúdo de lactato, sem alteração no conteúdo de piruvato. Portanto, os resultados do presente estudo sugerem que os tecidos de M. mactroides utilizam a anaerobiose para obtenção de energia durante a exposição ao Cu, conforme demonstrado no músculo pedal e hemolinfa. Apesar disso, a hemolinfa não é capaz de manter o nível de ATP nas condições experimentais testadas. 

Análise da expressão de APE1 após estresse oxidativo induzido em células proficientes e deficientes na via de reparo por excisão de nucleotídeos.

Riboflavin is a vitamin very important in aerobic organisms, as a precursor of many coenzymes involved in the electron transporter chain. However, after photosensitization of riboflavin with UV or visible light, it generates reactive oxygen species (ROS), which can oxidize the DNA. The repair of oxidative lesions on DNA occurs through the base excision repair pathway (BER), where APE1 endonuclease plays a central role. On the other hand, the nucleotide excision repair pathway (NER) repairs helix-distorting lesions. Recently, it was described the participation of NERproteins in the repair of oxidative damage and in stimulation of repair function fromAPE1. The aim of this research was to evaluate the cytotoxic effects of photosensitized riboflavin (RF*) in cells proficient and deficient in NER, correlating with APE1 expression. For this propose, the cells were treated with RF* and it was performed the cell viability assay, extraction of whole proteins, cells fractionation, immunoblotting, indirect immunofluorescence and analysis of polymorphisms of BER gens. The results evidenced that cells deficient in XPA and CSB proteins were more sensitive to RF*. However, XPC-deficient cells presented similar resistance to MRC5- SV cells, which is proficient in NER. These results indicate that XPA and CSB proteins have an important role on repair of oxidative lesions induced by RF*. Additionally, it was evidenced that single nucleotide polymorphisms (SNPs) in BER enzymes may influence in sensitivity of NER-deficient cell lines. Concerning the APE1 expression, the results showed that expression of this protein after treatment with RF* only changed in XPC-deficient cells. Though, it was observed that APE1 is recruited and is bound to chromatin in MRC5-SV and XPA cells after treatment with RF*. The results also showed the induction of DNA damage after treatment with RF*, through the analysis of-H2AX, since the treatment promoted an increase of endogenous levels of this phosphorylated protein, which acts signaling double strand-break on DNA. On the other hand, in XPC-deficient cells, regardless of resistance of RF*, the endogenous levels of APE1 are extremely reduced when compared with other cell lines and APE1 is not bound to chromatin after treatment with RF*. These results conclude that RF* was able to induce cell death in NERdeficient cells, where XPA and CSB cells were more sensitive when compared with MRC5-SV and XPC-deficient cells. This last result is potentially very interesting, since XPC-deficient cell line presents low levels of APE1. Additionally, the results evidenced that APE1 protein can be involved in the repair of oxidative damage induced by RF*, because APE1 is recruited and bound strongly to chromatin after treatment.