Decontamination of Mikania glomerata Leaves by Gamma Irradiation: Coumarin Determination by HPLC-DAD, Microbiological Control and Genotoxicological Studies.

Gamma irradiation as a decontaminating physical agent could be an important tool in the production chain of herbal medicines by improving the microbiological quality of raw materials and the safety of final products. This study was undertaken to investigate the genotoxic potential and eventual chemical modifications of a batch of Mikania glomerata raw material decontaminated by different doses of gamma irradiation (2.0, 3.5, and 5.0 kGy), using a cesium-137 source. DNA damage was assessed in vitro by agarose gel electrophoresis in regard to double-chain breaks of plasmid pUC 9.1 DNA and in vivo by micronucleus test in bone marrow cells of Wistar rats. Cytotoxicity in bone marrows was assessed by scoring polychromatic and normochromatic erythrocytes ratio. An HPLC-DAD method was adapted and validated for the enhancement of coumarin selectivity from the other matrix constituents. The microbial load was satisfactorily reduced, leading to sterilization at the highest dose. Genotoxic and cytotoxic effects were not increased in the in vitro and in vivo models. The concentration of coumarin and the chromatographic profiles of the hydroalcoholic plant extracts (ethanol 70% v/v) were not modified after such process. Therefore, this work suggests that gamma irradiation of M. glomerata raw material is suitable and safe for microbiological control purposes at the employed doses.

Apoptosis induction of cardiomyocytes and subsequent fibrosis after irradiation and neoadjuvant chemotherapy.

PURPOSE: Breast cancer treatments can induce important cardiovascular complications. The aim of this study was to evaluate cardiac alterations after irradiation and chemotherapy in an animal model. MATERIAL AND METHODS: Wistar rats were divided into three groups: Control, TC+ IR (received chemotherapy and irradiation) and IR (received only irradiation). After 5 months, echocardiography was performed, the animals were euthanized, and the left ventricle was analyzed using light microscopy techniques and Polymerase Chain Reaction (PCR). RESULTS: Echocardiography showed decreases in ejection fraction and cardiac output, in TC+ IR group. Both TC+ IR and IR showed reduced intramyocardial vessel-to-cardiomyocyte ratio, increased connective tissue, cardiomyocyte hypertrophy, increased numbers of apoptotic nuclei and increased Bax/Bcl2 expression. We also observed increased Transforming growth factor (TGF) beta 1 mRNA expression in both groups, but type 1 Procollagen expression was increased in TC+ IR group only. CONCLUSIONS: The study suggests that the induced cardiac remodelling begins with the reduction of intramyocardial vessels in the left ventricle tissue. The main consequence is the loss of cardiomyocytes through apoptosis, leading to the replacement of healthy tissue by fibrous tissue. It was observed that the damage caused by the combination of irradiation and chemotherapy induced functional alterations that did not occur when the animals were only irradiated.

MiR-34a is up-regulated in response to low dose, low energy X-ray induced DNA damage in breast cells.

BACKGROUND: MicroRNAs are non-coding RNAs involved in the regulation of gene expression including DNA damage responses. Low doses of low energy X-ray radiation, similar to those used in mammographic exams, has been described to be genotoxic. In the present work we investigated the expression of miR-34a; a well described p53-regulated miRNA implicated in cell responses to X-ray irradiation at low doses. METHODS: Non-cancerous breast cell line MCF-10A and cancerous T-47D and MCF-7 cell lines were submitted to a low-energy X-ray irradiation (ranging from 28-30 Kv) using a dose of 5 Gy. The expression level of miR-34a, let-7a and miR-21 was assessed by qRT-PCR at 4 and 24 hours post-irradiation. DNA damage was then measured by comet assay and micronuclei estimation in MCF-10A and MCF-7 cell lines, where an increase of miR-34a levels could be observed after irradiation. The rate of apoptotic cells was estimated by nuclear staining and fluorescence microscopy. These experiments were also performed at low doses (3; 12 and 48 mGy) in MCF-10A and MCF-7 cell lines. RESULTS: We have observed an increase in miR-34a expression 4 hours post-irradiation at 5 Gy in MCF-10A and MCF-7 cell lines while its level did not change in T-47D, a breast cancer cell line bearing non-functional p53. At low doses, miR-34a was up-regulated in non-tumoral MCF-10A to a higher extent as compared to MCF-7. MiR-34a levels decreased 24 hours post-irradiation. We have also observed DNA damage and apoptosis at low-energy X-ray irradiation at low doses and the high dose in MCF-10A and MCF-7 4 and 24 hours post-irradiation relative to the mock control. CONCLUSION: Low energy X-ray is able to promote DNA strand breaks and miR-34a might be involved in cell responses to low energy X-ray DNA damage. MiR-34a expression correlates with X-ray dose, time after irradiation and cell type. The present study reinforces the need of investigating consequences of low dose X-ray irradiation of breast cells.

DNA repair gene expression in biological tissues exposed to low-intensity infrared laser.

Special properties of laser light have led to its usefulness in many applications in therapy. Excitation of endogenous chromophores in biotissues and generation of free radicals could be involved in its biological effects. DNA lesions induced by free radicals are repaired by base excision repair pathway. In this work, we evaluated the expression of APE1 and OGG1 genes related to repair of DNA lesions induced by free radicals. Skin and muscle tissues of Wistar rats were exposed to low-intensity infrared laser at different fluences and frequencies. After laser exposition of 1 and 24 h, tissue samples were withdrawn for total RNA extraction, cDNA synthesis, and evaluation of APE1 and OGG1 gene expression by quantitative polymerase chain reaction. Data obtained show that laser radiation alters the expression of APE1 and OGG1 mRNA differently in skin and muscle tissues of Wistar rats depending of the fluence, frequency, and time after exposure. Our study suggests that low-intensity infrared laser affects expression of genes involved in repair of DNA lesions by base excision repair pathway.

Chemotherapy and radiation regimens to breast cancer treatment induce changes in mRNA levels of renin-angiotensin system related genes in cardiac tissue.

BACKGROUND AND AIM: Cardiovascular complications are one limitation of breast cancer treatment. The aim of the current study was to investigate whether the renin-angiotensin related genes could be altered by chemotherapy and radiotherapy, using a rat model. METHODS: Female rats were divided into three groups: control, chemotherapy + irradiation (TC+IR) and irradiation (IR). Molecular analyses of the left ventricle were performed five months after the end of treatment. The analyses evaluated the changes in mRNA levels of some renin-angiotensin system (RAS) related genes: angiotensinogen, renin, angiotensin-converting enzyme (ACE) and angiotensin II type 1 receptor (AT1) and vascular endothelial growth factor (VEGF), which can be related to ACE production, by RT-PCR. RESULTS: Renin was only observed in treated groups, TC+IR and IR, compared with the control group. ACE and VEGF levels were decreased in TC+IR (p<0.001) and in IR (p<0.001), and AT1 mRNA was higher in groups TC+IR (p<0.01) and IR (p<0.05) compared with the control group. CONCLUSION: Chemotherapy and irradiation can induce significant changes in some RAS related genes. These alterations are important to understand the pathways and consequences beyond cardiotoxicity induced by breast cancer treatments.

Up-regulation of angiotensin-converting enzyme and angiotensin II type 1 receptor in irradiated rats.

PURPOSE: To investigate changes in cardiac functional parameters and the cardiac expression of angiotensin-converting enzyme (ACE), angiotensin II type 1 receptor (AT1), procollagen type I (proc-I) and transforming growth factor-ß1 (TGF-ß1) in rats irradiated at heart. MATERIAL AND METHODS: Male Wistar rats were irradiated with a single dose of radiation (0, 5, 10 and 15 Gray [Gy]) delivered directly to the heart and the molecular evaluations were performed at various times post-irradiation (two days, 15 days and four months). The expression of ACE, AT1, proc-I and TGF-ß1 were analysed using Real Time-Polymerase Chain Reaction (RT-PCR) and/or Western blotting. Cardiac structural and functional alterations were investigated at the four-month time point by echocardiography and by quantitative methods (stereology). RESULTS: Rats irradiated with 15 Gy showed a modest reduction in the ejection fraction. Cardiac proc-I, TGF-ß1, ACE and AT1 were also measurably increased. CONCLUSIONS: Irradiated rat hearts show simultaneous elevations in renin-angiotensin system components AT1 and ACE and cardiac remodeling markers proc-I and TGF-ß1.