Emphysema induced by elastase alters the mRNA relative levels from DNA repair genes in acute lung injury in response to sepsis induced by lipopolysaccharide administration in Wistar rats.

Purpose/Aim of the study: Patients suffering from chronic obstructive pulmonary disease (COPD) in association with acute respiratory distress syndrome (ARDS) present oxidative stress in lung cells, with production of free radicals and DNA lesions in pulmonary and adjacent cells. Once the DNA molecule is damaged, a set of enzymatic mechanisms are trigged to preserve genetic code integrity and cellular homeostasis. These enzymatic mechanisms include the base and the nucleotide excision repair pathways, as well as telomere regulation. Thus, the aim of this work was to evaluate the mRNA levels from APEX1, ERCC2, TP53, and TRF2 genes in lung tissue from Wistar rats affected by acute lung injury in response to sepsis and emphysema. MATERIALS AND METHODS: Adult male Wistar rats were randomized into 4 groups (n = 6, for each group): control, emphysema, sepsis, and emphysema with sepsis. Pulmonary emphysema was induced by intratracheal instillation of elastase (12 IU/animal) and sepsis induced by intraperitoneal Escherichia coli lipopolysaccharide (LPS) injection (10 mg/kg). Lungs were removed, and samples were withdrawn for histological analysis and total RNA extraction, cDNA synthesis, and mRNA level evaluation by real time quantitative polymerase chain reaction. RESULTS: Data show acute lung injury by LPS and emphysema by elastase and that APEX1, ERCC2, TP53, and TRF2 mRNA levels are increased significantly (p < 0.01) in emphysema with sepsis group. CONCLUSION: Our results suggest that alteration in mRNA levels from DNA repair and genomic stability could be part of cell response to acute lung injury in response to emphysema and sepsis.

Low-level infrared laser modulates muscle repair and chromosome stabilization genes in myoblasts.

Infrared laser therapy is used for skeletal muscle repair based on its biostimulative effect on satellite cells. However, shortening of telomere length limits regenerative potential in satellite cells, which occurs after each cell division cycle. Also, laser therapy could be more effective on non-physiologic tissues. This study evaluated low-level infrared laser exposure effects on mRNA expression from muscle injury repair and telomere stabilization genes in myoblasts in normal and stressful conditions. Laser fluences were those used in clinical protocols. C2C12 myoblast cultures were exposed to low-level infrared laser (10, 35, and 70 J/cm(2)) in standard or normal (10 %) and reduced (2 %) fetal bovine serum concentrations; total RNA was extracted for mRNA expression evaluation from muscle injury repair (MyoD and Pax7) and chromosome stabilization (TRF1 and TRF2) genes by real time quantitative polymerization chain reaction. Data show that low-level infrared laser increases the expression of MyoD and Pax7 in 10 J/cm(2) fluence, TRF1 expression in all fluences, and TRF2 expression in 70 J/cm(2) fluence in both 10 and 2 % fetal bovine serum. Low-level infrared laser increases mRNA expression from genes related to muscle repair and telomere stabilization in myoblasts in standard or normal and stressful conditions.

Low-intensity red and infrared lasers affect mRNA expression of DNA nucleotide excision repair in skin and muscle tissue.

Lasers emit light beams with specific characteristics, in which wavelength, frequency, power, fluence, and emission mode properties determine the photophysical, photochemical, and photobiological responses. Low-intensity lasers could induce free radical generation in biological tissues and cause alterations in macromolecules, such as DNA. Thus, the aim of this work was to evaluate excision repair cross-complementing group 1 (ERCC1) and excision repair cross-complementing group 2 (ERCC2) messenger RNA (mRNA) expression in biological tissues exposed to low-intensity lasers. Wistar rat (n = 28, 4 for each group) skin and muscle were exposed to low-intensity red (660 nm) and near-infrared (880 nm) lasers at different fluences (25, 50, and 100 J/cm(2)), and samples of these tissues were withdrawn for RNA extraction, cDNA synthesis, and gene expression evaluation by quantitative polymerase chain reaction. Laser exposure was in continuous wave and power of 100 mW. Data show that ERCC1 and ERCC2 mRNA expressions decrease in skin (p < 0.001) exposed to near-infrared laser, but increase in muscle tissue (p < 0.001). ERCC1 mRNA expression does not alter (p > 0.05), but ERCC2 mRNA expression decreases in skin (p < 0.001) and increases in muscle tissue (p < 0.001) exposed to red laser. Our results show that ERCC1 and ERCC2 mRNA expression is differently altered in skin and muscle tissue exposed to low-intensity lasers depending on wavelengths and fluences used in therapeutic protocols.

Effect of laser therapy on DNA damage.

BACKGROUND AND OBJECTIVE: Whereas the biostimulative effect on tissues using low intensity laser therapy for treating many diseases has been described, the photobiological basis and adverse effects are not well understood. The aim of this study, using experimental models, is to observe the combined effect of physical damage (laser) and a chemical agent (hydrogen peroxide) on Escherichia coli cultures and bacterial plasmids. MATERIALS AND METHODS: Survival of E. coli AB1157 (wild type) and BW9091 (xth(-)) cultures were used as an experimental model to assess the effect of agents on DNA, also agarose gel electrophoretic profile of bacterial plasmids for studying single and double strand breaks in DNA exposed to laser irradiation and in DNA pre-exposed to laser and subsequently incubated with hydrogen peroxide. RESULTS: Data indicate low intensity laser: (i) did not alter the survival of E. coli cultures, (ii) pre-exposure had a protective effect against lethal action of hydrogen peroxide on E. coli cultures, and (iii) did not alter the electrophoretic profile and action of hydrogen peroxide on plasmids. This suggests that low intensity therapeutic red laser doses at different emission modes induces sub-lethal effects on E. coli wild type and exonuclease III mutant cultures inducing protective mechanisms against lethal action of hydrogen peroxide. Laser action on bacterial plasmids is related to lesions other than single or double DNA strands breaks. CONCLUSIONS: This study shows a protective effect or DNA repair mechanism induction by pre-exposure to low intensity red laser on the lethal action of oxidant agents and, therefore, laser therapy protocol should consider fluencies, wavelength and tissue conditions before beginning treatment.

Biological effects of an aqueous extract of Salix alba on the survival of Escherichia coli AB1157 cultures submitted to the action of stannous chloride.

Stannous chloride (SnC12) is used in nuclear medicine as a reducing agent to obtain technetium-99m-radiopharmaceuticals. It have been reported that natural products might reduce the genotoxic and cytotoxic effects related to SnC12. This work evaluated the biological effects of an aqueous extract of Salix alba on the survival of Escherichia coli (E. coli) AB1157 (wild type) cultures submitted to the action of SnC12. E. coli AB1157 cultures (exponential growth phase) were collected by centrifugation, washed and resuspended in 0.9% NaCl. Samples were incubated in water bath shaker with: (a) SnC12 (25 microg/ml), (b) Salix alba extract(11.6 mg/ml) and (c) SnC12 (25 microg/ml) + Salix alba extract (11.6 mg/ml). Incubation with 0.9% NaCl was also carried out (control). At 60 min intervals, aliquots were withdrawn, diluted, spread onto Petri dishes with solid LB medium and incubated overnight. The colonies formed were counted and the survival fractions calculated. The extract was not able to protect the E. coli cultures against the lesive action of SnC12. The extract also did not interfere with the survival of the cultures. It suggested that the substances present in the Salix alba aqueous extract did not interfere strongly with cellular metabolism and did not alter the survival fractions of E. coli AB 1157. It is speculated that this extract cannot interfere with the generation of free radicals, the possible main agent responsible for SnC12 lesive action.

In vitro and in vivo studies of an aqueous extract of Matricaria recutita (German chamomile) on the radiolabeling of blood constituents, on the morphology of red blood cells and on the biodistribution of the radiopharmaceutical sodium pertechnetate.

BACKGROUND: Natural products might alter the labeling of blood constituents with technetium-99m ((99m)Tc) and these results may be correlated with modifications of the shape of the red blood cells (RBC). The biodistribution of radiopharmaceuticals can be also altered. OBJECTIVE: This investigation aimed to determine biological effects of an aqueous extract of chamomile (CE). MATERIALS AND METHODS: To study the effect of the CE on the labeling of blood constituents with (99m)Tc, in vitro and in vivo assays were performed. The effect of the CE on the morphology of RBC was observed under light microscope. The images were acquired, processed, and the perimeter/area ratio of the RBC determined. To analyze the effect of the CE on biodistribution of the sodium pertechnetate (Na(99m)TcO4) in Wistar rats, these animals were treated or not with a CE. Na(99m)TcO4 was injected, the rats were sacrificed, the organs were removed, weighted and percentage of radioactivity/gram calculated. RESULT: In the in vitro experiment, the radioactivity on blood cells compartment and on insoluble fractions of plasma was diminished. The shape and the perimeter/area ratio of the RBC were altered in in vitro assays. An increase of the percentage of radioactivity of Na(99m)TcO4 was observed in stomach after in vivo treatment. CONCLUSION: These results could be due to substances of the CE or by the products of the metabolism of this extract in the animal organism. These findings are examples of drug interaction with a radiopharmaceutical, which could lead to misdiagnosis in clinical practice with unexpected consequences.

Biological effects of an aqueous extract of Salix alba on the survival of Escherichia coli AB1157 cultures submitted to the action of stannous chloride

Stannous chloride (SnC12) is used in nuclear medicine as a reducing agent to obtain technetium-99m-radiopharmaceuticals. It have been reported that natural products might reduce the genotoxic and cytotoxic effects related to SnC12. This work evaluated the biological effects of an aqueous extract of Salix alba on the survival of Escherichia coli (E. coli) AB1157 (wild type) cultures submitted to the action of SnC12. E. coli AB1157 cultures (exponential growth phase) were collected by centrifugation, washed and resuspended in 0.9 percentNaCl. Samples were incubated in water bath shaker with: (a) SnC12 (25mg/ml), (b)Salix alba extract(11.6mg/ml) and (c)SnC12(25mg/ml) + Salix alba extract (11.6mg/ml). Incubation with 0.9 percent NaCl was also carried out (control). At 60 min intervals, aliquots were withdrawn, diluted, spread onto Petri dishes with solid LB medium and incubated overnight. The colonies formed were counted and the survival fractions calculated. The extract was not able to protect the E. coli cultures against the lesive action of SnC12. The extract also did not interfere with the survival of the cultures. It suggested that the substances present in the Salix alba aqueous extract did not interfere strongly with cellular metabolism and did not alter the survival fractions of E. coli AB 1157. It is speculated that this extract cannot interfere with the generation of free radicals, the possible main agent responsible for SnC12 lesive action.

Influence of an aqueous extract of Hypericum perforatum (Hypericin) on the survival of Escherichia coli AB1157 and on the electrophoretic mobility of pBSK plasmid DNA / Influência de um extrato aquoso de Hypericum perforatum (Hipericina) na sobreviência de Escherichia coli AB1157 e na mobilidade eletroforética de DNA plasmidial pBSK

Hiperico (Hypericum perforatum or St John's worth) has been widely used as an herbal medicine to treat depression. Hypericin is the main chemical compound of hiperico. Stannous chloride (SnCl2) is the most used reducing agent in nuclear medicine. The aim of this work was to verify the effect of a hiperico extract on the survival of Escherichia coli AB1157 and on the plasmid DNA topology. Exponentially E. coli AB1157 cultures were incubated with SnCl2 in the presence or absence of hypericin. Aliquots were spread onto Petri dishes containing solidified rich medium, the colonies units were counted after overnight and the survival fraction was calculated. Plasmid DNA samples were incubated with SnCl2 in presence or absence of hypericin extract during 40 minutes, 0.8 percent agarose gel electrophoresis was performed, the gel was stained with ethidium bromide and the plasmid topological forms (bands) were visualized. The results revealed that hiperico extract is neither capable of altering the survival of E. coli cells nor the plasmid DNA topology but it may have protected these cells against the SnCl2 action. The data suggest absence of cytotoxic and genotoxic effects of the aqueous hiperico extract and a protective effect on E. coli cells against the action of SnCl2.

Hipérico (Hypericum perforatum or St John's worth) tem sido usado como uma planta medicinal para tratar a depressão. Hipericina é o principal componente do hipérico. O cloreto estanoso (SnCl2) é o agente redutor mais utilizado em medicina nuclear. O objetivo desse trabalho foi verificar o efeito de um extrato de hipérico na sobrevivência de Escherichia coli AB1157 e na topologia do DNA plasmidial. Culturas de E. coli AB1157, em fase exponencial, foram incubadas com SnCl2 na presença ou ausência de hipericina. Alíquotas foram espalhadas em placas de Petri contendo meio sólido, as unidades formadoras de colônias foram contadas após incubação e as frações de sobrevivência calculadas. DNA plasmidial foi incubado com SnCl2 na presença ou ausência de hipericina durante 40 minutos, eletroforese em gel de agarose a 0,8 por cento foi realizada, o gel foi corado com brometo de etídio e as formas (bandas) topológicas do plasmídeo visualizadas. Os resultados revelaram que o extrato de hipérico não foi capaz de alterar a sobrevivência da cultura de E. coli e a topologia do DNA plasmidial, mas protegeu as bactérias contra a ação do SnCl2. Os resultados sugerem ausência de efeitos citotóxicos e genotóxicos do extrato aquoso do hipérico e um efeito protetor nas células de E. coli contra a ação do SnCl2.

Osmotic and morphological effects on red blood cell membrane: action of an aqueous extract of Lantana camara / Efeitos osmótico e morfológico na membrana de hemácias: ação de um extrato aquoso de Lantana camara

The Lantana camara ("cambara de espinho") leaves infusions are used popularly in some countries to treat gastrointestinal diseases. Osmotic fragility assay and morphometric analysis have been used to verify the interaction of drugs with the membrane of red blood cells (RBC). The aim of this work was to evaluate the effects of an aqueous extract of Lantana camara on the osmotic fragility and on the morphology of RBC. Blood samples were treated with extract of Lantana camara (10 mg/mL), osmotic fragility assay and morphological analysis were carried out. In the presence of the extract, the data obtained indicated (i) a significant (p < 0.05) increase of hemolysis and (ii) modifications on the morphology of RBC. These effects of the Lantana camara may be associated with some pharmacological properties of the chemical compounds of this studied extract.

As infusões de folhas de Lantana camara (cambara-de-espinho) são usadas popularmente em alguns países para tratar doenças gastrointestinais. O experimento de fragilidade osmótica e a análise morfométrica têm sido usados para verificar a interação de drogas com a membrana de hemácias. O objetivo deste trabalho foi avaliar os efeitos de um extrato aquoso de Lantana camara na fragilidade osmótica e na morfologia de hemácias. Amostras de sangue foram tratadas com extrato de Lantana camara (10 mg/mL), o ensaio de fragilidade osmótica e a análise morfológica foram realizadas. Na presença do extrato, os dados obtidos indicaram (i) um aumento significativo (p < 0,05) da hemólise e (ii) modificações na morfologia das hemácias. Estes efeitos da Lantana camara poderiam estar associados com algumas propriedades farmacológicas de compostos químicos do extrato estudado.

Effects of a tomato (Solanum lycopersicum) extract on the labeling of blood constituents with technetium-99m / Efeitos de um extrato de tomate (Solanum lycopersicum) na marcação de constituintes sangüíneos com tecnécio-99m

O tomate (Solanum lycopersicum) é o segundo vegetal mais produzido e consumido no mundo, tendo sido indicado para prevenção e tratamento de câncer, asma e arteriosclerose. Constituintes sangüíneos marcados com radionuclídeos têm sido usados em procedimentos na medicina nuclear. Dados têm mostrado que alimentos e drogas podem alterar a marcação de constituintes sangüíneos com tecnécio-99m (99mTc). Este estudo avaliou a influência de um extrato de tomate neste procedimento de radiomarcação. Sangue heparinizado (Wistar rats) foi incubado in vitro com diferentes concentrações de um extrato de tomate e a marcação com 99mTc foi realizada. Plasma (P) e células sangüíneas (CS) foram separadas permitindo o isolamento das frações solúvel (SF-P/SF-CS) e insolúvel (IF-P/IF-CS) por precipitação e centrifugação. A radioatividade nos constituintes sangüíneos (P, CS, IF-P, SF-P, IF-CS e SF-CS) foi determinada e a porcentagem de radioatividade ( por centoATI), calculada. O extrato de tomate usado, nas maiores concentrações (2,00 e 4,00g/mL), reduziu significativamente (p < 0,05) a por centoATI na IF-P, embora este extrato não tenha modificado a radiomarcação da CS e fixação da radioatividade na IF-CS. Em conclusão, nossos dados sugerem que os compostos químicos presentes no extrato aquoso de tomate teriam algumas propriedades capazes de alterar a fixação do 99mTc nas proteínas plasmáticas.

Tomato (Solanum lycopersicum) is the second most produced and consumed vegetable in the world. It has been indicated in the prevention and treatment of cancer, asthma and atherosclerosis. Blood constituents labeled with radionuclides have been used in procedures in nuclear medicine. Data have shown that food and drugs can alter the labeling of blood constituents with technetium-99m (99mTc). This study evaluated the influence of a tomato extract on this radiolabeling procedure. Heparinized blood (Wistar rats) was incubated in vitro with different concentrations of a tomato extract and 99mTc-labeling was performed. Plasma (P) and blood cells (BC) were separated following soluble (SF-P/SF-BC) and insoluble (IF-P/IF-BC) fractions isolation by precipitation and centrifugation. The radioactivities on blood constituents (P, BC, IF-P, SF-P, IF-BC and SF-BC) were determined and the percentage of radioactivity ( percentATI) was calculated. The tomato extract used at the highest concentrations (2.00 and 4.00g/mL), reduced significantly (p < 0.05) the percentATI in IF-P, although this extract did not modify the radiolabeling on BC, neither the radioactivity fixation on IF-BC. In conclusion, our data suggest that the chemical compounds present in the aqueous tomato extract could have some properties capable of change the fixation of 99mTc on plasma proteins.

A Chrysobalanus icaco extract alters the plasmid topology and the effects of stannous chloride on the DNA of plasmids

Chrysobalanus icaco (C. icaco) leaves are used in folk medicine (known as Abajeru in Brazil) to control the glycaemia in diabetic patients. Stannous chloride (SnCl2) is a powerful reducing agent used for different purposes and presents cytotoxic and genotoxic effects. The aim of this work was to investigate the effect of an aqueous C. icaco extract on the plasmid DNA topology and on the effects of the stannous chloride on DNA plasmid. Plasmid pBSK was incubated with a C. icaco extract in the presence or absence of SnCl2 (200 mg/mL), after that, the agarose gel electrophoresis procedure was carried out. Plasmid incubated only SnCl2 was used as positive control and, as negative control, plasmid incubated with Tris buffer. The gels were stained with ethidium bromide, DNA bands were semiquantified by densitometry. The data showed that C. icaco extract alters the electrophoretic profile and decreases significantly (p < 0.05) the effect of SnCl2 on plasmid DNA. The results obtained in this work could indicate a dose-dependent protective action and a genotoxic effect of C. icaco extract on plasmid DNA.

Folhas de Chrysobalanus icaco (C. icaco) são usadas na medicina popular (conhecido como Abajeru no Brasil) para controlar a glicemia em pacientes diabéticos. Cloreto estanoso (SnCl2) é um agente redutor potente usado para diferentes propostas e apresenta efeitos citotóxico e genotóxico. O objetivo deste trabalho foi investigar os efeitos de um extrato aquoso de C. icaco na topologia de DNA plasmidial e nos efeitos do cloreto estanoso sobre o DNA plasmidial. Plasmídios pBSK foram incubados com um extrato de C. icaco na presença ou ausência do SnCl2 (200 mg/mL), em seguida, o procedimento de eletroforese em gel de agarose foi realizado. Plasmídios incubados somente com SnCl2 foram usados como controle positivo e, como controle negativo, plasmídios incubados com tampão Tris. Os géis foram corados com brometo de etídio e as bandas de DNA foram semiquantificadas por densitometria. Os dados mostraram que o extrato de C. icaco altera o perfil eletroforético e diminui significativamente (p < 0,05) os efeitos do SnCl2 sobre DNA plasmidial. Os resultados obtidos neste trabalho indicam uma ação protetora dependente da dose e um efeito genotóxico de extrato de C. icaco sobre o DNA plasmidial.

Assessment of effects of a formula used in the traditional Chinese medicine (Buzhong Yi Qi Wan) on the morphologic and osmotic fragility of red blood cells

Buzhong Yi Qi Wan (BYQW) is a combination of some medicinal herbs widely used in traditional Chinese medicine to treat blood, spleen and stomach disorders. Morphometric analysis and osmotic fragility assay have been used to evaluate changes on membrane integrity of red blood cells. The aim of this work was to evaluate the effect of an aqueous BYQW extract on the morphology and osmotic fragility of red blood cells. Blood samples were treated with BYQW extract, quantitative/qualitative morphological analysis and osmotic fragility assay were carried out against control groups treated with saline. The data obtained indicated no modification on morphology but osmotic fragility assay suggested a significant (p<0.05) increasing of hemolysis in red blood cells isolated from blood treated with aqueous BYQW extract. In conclusion, the aqueous BYQW extract could affect the membrane integrity decreasing the osmotic resistance but without altering the shape of red blood cells.

Buzhong Yi Qi Wan (BYQW) é uma combinação de algumas ervas medicinais amplamente usada na medicina tradicional chinesa para tratar o sangue, baço e desordens do estômago. A análise morfométrica e o ensaio de fragilidade osmótica têm sido usados para avaliar alterações na integridade da membrana de hemácias. O objetivo deste trabalho foi avaliar os efeitos de um extrato aquoso de BYQW na morfologia e na fragilidade osmótica de hemácias. Amostras sangüíneas foram tratadas com o extrato de BYQW, análise morfológica quantitativa/qualitativa e o ensaio de fragilidade osmótica foram realizados e comparados com grupo controle tratado com salina. Os dados obtidos indicaram ausência de modificações na morfologia, mas o ensaio de fragilidade osmótica sugeriu um aumento significativo (p<0,05) da hemólise em hemácias isoladas de sangue tratado com extrato aquoso de BYQW. Em conclusão, o extrato aquoso de BYQW poderia afetar a integridade da membrana diminuindo a resistência osmótica sem alterar a forma das hemácias.