Genotoxic damage to glioblastoma cells treated with 6 MV X-radiation in the presence or absence of methoxy estradiol, IUDR or topotecan

To explore the cumulative genotoxic damage to glioblastoma [GBM] cells, grown as multicellular spheroids, following exposure to 6 MV X-rays [2 Gy, 22 Gy] with or without, 2- methoxy estradiol [2ME2], iododeoxyuridine [IUDR] or topotecan [TPT], using the Picogreen assay. The U87MG cells cultured as spheroids were treated with 6 MV X-ray using linear accelerator. Specimens were divided into five groups and irradiated using X-ray giving the dose of 2 Gy after sequentially incubated with one of the following three drug combinations: TPT, 2-ME2/TPT, IUDR/TPT or 2ME2/IUDR/TPT. One specimen was used as the irradiated only sample [R]. The last group was also irradiated with total dose of 22 Gy [each time 2 Gy] of 6 MV X-ray in 11 fractions and treated for three times. DNA damage was evaluated using the Picogreen method in the experimental study. R/TPT treated group had more DNA damage [double strand break [DSB]/single strand break [SSB]] compared with the untreated group [P<0.05]. Moreover the R/TPT group treated with 2ME2 followed by IUDR had maximum DNA damage in spheroid GBM indicating an augmented genotoxicity in the cells. The DNA damage was induced after seven fractionated irradiation and two sequential treatments with 2ME2/IUDR/TPT. To ensure accuracy of the slope of dose response curve the fractionated radiation was calculated as 7.36 Gy with respect to alpha/beta ratio based on biologically effective dose [BED] formulae. Cells treated with 2ME2/IUDR showed more sensitivity to radiation and accumulative DNA damage. DNA damage was significantly increased when GBM cells treated with TPT ceased at S phase due to the inhibition of topoisomerase enzyme and phosphorylation of Chk1 enzyme. These results suggest that R/TPT-treated cells increase sensitivity to 2ME2 and IUDR especially when they are used together. Therefore, due to an increase in the level of DNA damage [SSB vs. DSB] and impairment of DNA repair machinery, more cell death will occur. This in turn may improve the treatment of GBM

comparison of anticancer activity of thymoquinone and nanothymoquinone on human breast adenocarcinoma

Cancer is one of the main causes of mortality in the world which is created by the effect of enviromental physico-chemical mutagen and carcinogen agents. The identification of new cytotoxic drugs with low side effects on immune system has developed as important area in new studies of pharmacology. Thymoquinone [TQ], derived from the medicinal spice Nigella sativa [also calledt black cumin] exhibit anti-inflammatory and anti-cancer activities. In this study we employed nanogel-based nanoparticle approach to improve upon its effectiveness. Myristic acid-chitosan [MA-chitosan] nanogels were prepared by the technique of selfassembly. Thymoquinone was loaded into the nanogels. The surface morphology of the prepared nanoparticles was determined using SEM and TEM. The other objective of this study was to examine the in-vitro cytotoxic activity of cell death of Thymoquinone and nanothymoquinone on human breast adenocarcinoma cell line [MCF7]. Cytotoxicity and viability of Thymoquinone and nanothymoquinone were assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide [MTT] and dye exclusion assay. Transmission electron microscopy confirmed the particle diameter was between 150 to 200 nm. Proliferation of MCF7 cells was significantly inhibited by Thymoquinone and nanothymoquinone in a concentration-dependent manner in defined times. There were significant differences in IC50 Thymoquinone and nanothymoquinone. TQ-loaded nanoparticles proved more effective compared to TQ solution. The high drug-targeting potential and efficiency demonstrates the significant role of the anticancer properties of TQ-loaded nanoparticles

Expression and characterization of bacterial organophosphorus hydrolase in pichia pastoris with the intent to degrade organophosphate neurotoxins

Organophosphorus hydrolase [OPH] is a homodimeric enzyme that can hydrolyze phosphoester bonds and reduce the toxicity of organophosphorus compounds. This makes OPH a suitable element for the biodegradation of these compounds. We successfully cloned the OPH gene from Pseudomonas diminuta, after optimization for Pichia pastoris, into a yeast expression vector [pPICZlphaB]. After transformation and induction of recombinant yeasts, the expressed enzyme was investigated for its biochemical and kinetical parameters. The enzyme was purified 7.49-fold to a specific activity of 0.421×10[3] U/mg protein from the supernatant with a yield of 33%. The purified enzyme was able to degrade organophosphates. It had an optimal activity and stability up to 50°C, and a pH range of 7.0-10.0. The enzyme had a Km of 45.96 µM and a Vmax of 11.23 microM/min [421 microM/min/mg] for paraoxon as a substrate. This enzyme was sensitive to divalent cations and inactivated by denaturing compounds such as SDS. The molecular mass of the purified enzyme as estimated by SDS-PAGE analysis was approximately 40 kDa. In this study, the purified enzyme effectively hydrolyzed paraoxon, an organophosphorus compound. The activity and stability of this enzyme at high temperatures and pH, and low Km in comparision with bacterial isolates could make it an attractive biocatalyst for applied bioremediation and biosensing

Endothelial dysfunction in experimental atherosclerosis in the rabbit with extraction of instantaneous changes in the arterial wall

In this study, we used a new computerized analytical method for the measurement of the endothelial function in sequential ultrasound images and compared it with histological studies, using the abdominal aorta in normal and atherosclerotic rabbits. Six rabbits received a standard rabbit chow as the normal group and the other 6 rabbits were fed a high cholesterol diet for four weeks as the atherosclerotic group. B-mode images of the abdominal aorta with 46 frames per second were saved over three cardiac cycles at baseline and during acetylcholine or nitroglycerin drug infusion in the normal and atherosclerotic rabbits. In order to evaluate endothelial-dependent relaxation, acetylcholine-mediated dilation [AMD] was measured during the infusion of acetylcholine at a rate of 0.5 microg/kg/min and endothelial-independent relaxation was evaluated by measuring nitroglycerine-mediated dilation [NMD] during the infusion of nitroglycerin at a rate of 5 microg/kg/min. In addition, the ultrasonic evaluation was confirmed by histopathological evaluation of the abdominal aorta. Significant differences in AMD were detected between the normal and the four-week cholesterol-fed rabbits [p value < 0.05], whereas there were no significant differences in NMD between the two groups [p value > 0.05]. No microscopic intimal lesions were seen in the normal rabbits, but intimal thickening was observed in the histological studies in the four week cholesterol-fed rabbits. Additionally, the total cholesterol, triglycerides, low-density lipoprotein cholesterol, and high density lipoprotein cholesterol levels were remarkably increased in the sera of the four-week cholesterol-fed rabbits [p value < 0.05]. A new automatic method can help accurately evaluate the endothelial function in normal and hypercholesterolemic rabbits.

Developing a rabbit model of neointimal stenosis and atherosclerotic fibrous plaque rupture

A precise understanding of the mechanism of human neointimal stenoses and atherosclerotic fibrous plaques, which give rise to thromboses in vital arteries, requires a suitable animal model that would mimic the same characteristics well. We developed a rabbit model of neointimal stenosis and fibrotic plaque rupture in the carotid artery to visualize the lesion progress and to characterize the lesion types according to the American Heart Association classification. Twenty-eight healthy male New Zealand white rabbits were randomly divided into two groups: The rabbits in group A [n = 14] consumed a standard chow diet, and those in group B [n = 14] were injured via perivascular cold injury using liquid nitrogen at the right common carotid artery before being fed a high cholesterol diet [1.5%] for eight weeks. Plasma lipid evaluation was performed before the sacrificing of the rabbits. At the end of every week, at least 1 rabbit from group B was sacrificed for an analysis of lesion histopathology and calculation of the area ratios of the intima to media. The plasma lipid level in group B was significantly higher than that in group A [p value < 0.05]. The histopathological results revealed atherosclerosis characteristics such as endothelial layer destruction, fatty streaks and lipid-containing macrophages [foam cells] formation in the intima and media layers, extracellular lipid collections, smooth muscle cells proliferation and migration, neointima formation, intima thickening and deformation, fibrotic plaque formation, and finally plaque rupture. Statistical analysis revealed a significant increase in the intima-to-media ratio at the end of the eighth week [6.41 +/- 0.27, p value < 0.05]. We successfully developed a rabbit model of neointimal stenosis and atherosclerotic fibrous connective tissue plaque rupture, which is not only quickly and easily reproducible and inexpensive but also without mortality. The merits of our model render the evaluation of neointimal stenoses and fibrotic plaques and their treatment strategies more feasible in humans

Factors associated with secondhand smoke exposure in infants

Determining the factors associated with secondhand smoke [SHS] exposure in children provides valuable information for smoking control strategies. This study aimed to assess factors related to SHS exposure in infants based on urinary cotinine measures. A cross-sectional analysis of the data that were collected as part of the randomized controlled trial was conducted. Participants were 130 smoking households with children under the age of 1 year attending a health care center in southern Tehran. Eligible parents consented to participate in this study and completed a questionnaire including demographic data, questions regarding smoking at home, smoking status and Fagerstrom test through face-to-face interview. The Infants' urinary cotinine level was measured using gas chromatography, adjusted with urinary creatinine level and reported as cotinine [ng]/ creatinine [mg]. Factors related to infants' SHS exposure were assessed using the multivariate logistic regression model based on standard cut-point [30 ng of urinary cotinine/mg creatinine]. The final multivariate logistic regression model showed that social status [p=0.002], home smoking restriction [p=0.05] and the infant's age [p=0.01] were associated with the infants' SHS exposure determined based on urinary cotinine levels. These results support the influence of social status, home smoking restriction and infant's age on the exposure of infants to SHS

Estimation of atlatoxin-albumin adduct in serum sample by optimized HPLC-fluorescence

With consideration of lethal effects of aflatoxins specially Bl on human health. Estimation of aflatoxin-albumin adduct, as an important marker of aflatoxin exposure, seems essential. The aim of this study is optimization of HPLC-fluorescence method for measurement of this important marker in blood serum. In this study, blood serum of three groups of rats as A] positive controls [treated with AFB1], B] negative controls [without treatment] and standard rats [treated with radiolabeled AFBl] were used. After albumin isolation using ammunium sulphate and acetic acid, purity of albumin was tested by SDS-PAGE electrophoresis and albumin concentration was quantified by bradford method. Then albumin was hydrolysed by pronase and aflatoxin bound to albumin was released as aflatoxin-lysine. Pronase was precipitated and albumin was digested by aceton in cold, the volume of supernatant was reduced by freeze-drier and injected into HPLC system. Aflatoxin was quantified in comparison to standard rats samples. The purity of this isolated albumin was confirmed by SDS-PAGE electrophoresis. Albumin concentration in positive, negative and standard samples were 10, 13 and 12.5 mg/ml, respectively. Detection limit [20 pg/mg Alb] for measurement of aflatoxin was determined by HPLC method, specificity and sensitivity of method were 92% and 100% respectively. The mean concentration of AF-Alb adducts in serum of positive control rats was 10 ng/mg Alb and the reproducibility of the method after several repeat was very good. In this study, for AF-Alb adduct quantification by HPLC method, mobile phase, I percentage of solvents and run time were changed and the affinity chromatography before HPLC, was deleted. Therefor HPLC- fluorescence which is a precise and specific method, and since it is fast, highly reproducible and cost effective, also with improvement made, could easily be used for the quantification of this important marker in serum