The impact of autophagy on cell death modalities in CRL-5876 lung adenocarcinoma cells after their exposure to γ-rays and/or erlotinib

In most patients with lung cancer radiation treatment is used either as single agent or in combination with radiosensitizing drugs. However, the mechanisms underlying combined therapy and its impact on different modes of cell death have not yet been fully elucidated. We aimed to examine effects of single and combined treatments with γ-rays and erlotinib on radioresistant CRL-5876 human lung adenocarcinoma cells with particular emphasis on cell death. CRL-5876 cells were treated with γ-rays and/or erlotinib and changes in cell cycle, DNA repair dynamics, ultrastructure, nuclear morphology and protein expression were monitored at different time points. To reveal the relationship between types of cell death that arise after these treatments, autophagy was blocked with chloroquine. We found that higher dose of γ-rays causes G2/M arrest while adding of erlotinib to this treatment decreases the number of cells in S phase. Impact of erlotinib on kinetics of disappearance of irradiation-induced DNA double strand breaks is reflected in the increase of residual γ-H2AX foci after 24 h. γ-rays provoke cytoprotective autophagy which precedes development of senescence. Erlotinib predominantly induces apoptosis and enlarges the number of apoptotic cells in the irradiated CRL-5876 cells. Chloroquine improved cytotoxicity induced by radiation and erlotinib, increased apoptosis and decreased senescence in the CRL-5876 cells. The results obtained on CRL-5876 cells indicate significant radiosensitizing effect of erlotinib and suggest that chloroquine in the combination with the above treatments may have an additional antitumor effect in lung adenocarcinoma.     

Modulation of diabetes-related liver injury by the HMGB1/TLR4 inflammatory pathway

Chronic inflammation plays an essential role in the development of diabetic complications. Understanding the molecular mechanisms that support inflammation is a prerequisite for the design of novel anti-inflammatory therapies. These would take into consideration circulating levels of cytokines and damage-associated molecular patterns (DAMPs) that include the high mobility group box 1 (HMGB1) protein which, in part, promotes the inflammatory response through TLR4 signaling. The liver, as the source of circulating cytokines and acute-phase proteins, contributes to the control of systemic inflammation. We previously found that liver injury in streptozotocin-induced diabetic rats correlated with the level of oxidative stress, increased expression of HMGB1, and with the activation of TLR4-mediated cell death pathways. In the present work, we examined the effects of ethyl pyruvate (EP), an inhibitor of HMGB1 release/expression, on the modulation of activation of the HMGB1/TLR4 inflammatory cascade in diabetic liver. We observed that increased expression of inflammatory markers, TNF-α, IL-6, and haptoglobin in diabetic liver was associated with increased HMGB1/TLR4 interaction, activation of MAPK (p38, ERK, JNK)/NF-κB p65 and JAK1/STAT3 signaling pathways, and with decreased expression of Nrf2-regulated antioxidative enzymes. The reduction in HMGB1 expression as the result of EP administration reduced the pro-inflammatory activity of HMGB1 and exerted a protective effect on diabetic liver, which was observed as improved liver histology and antioxidant and inflammatory statuses. Our results suggest that prevention of HMGB1 release and blockage of the HMGB/TLR4 axis represents a potentially effective therapeutic strategy aimed at ameliorating diabetes-induced inflammation and ensuing liver injury.     

Chemical sympathectomy and diurnal fluctuations of noradrenaline calorigenic action in the rat

In the rat, in which a diurnal fluctuation of the sensitivity to noradrenaline was previously found, the effect of injected 6-hydroxydopamine (6-OHDA) was investigated. The heat production and catecholamines contents in the interscapular brown adipose tissue, heart and adrenals were measured. Chemical sympathectomy induces a disappearance of diurnal fluctuation in the sensitivity to injected noradrenaline. In these animals a lower capacity for heat production was found. However, a significant calorigenic effect of injected noradrenaline in 6-OHDA-treated animals was still present. In sympathectomized animals a depletion of noradrenaline from interscapular brown adipose tissue and the heart was observed. Besides, a change in adrenaline/noradrenaline ratio was found in the adrenals.     

The effects of cold acclimation and nitric oxide on antioxidative enzymes in rat pancreas

Alterations of pancreatic antioxidative defense (AD) and possible nitric oxide (NO) role in AD organization of adult rats receiving l-arginine.HCl (2.25%) or N(omega)-nitro-l-arginine methyl ester (L-NAME.HCl, 0.01%) as drinking liquids and maintained at room (22+/-1 degrees C) or low (4+/-1 degrees C) temperature for 45 days were studied. For that purpose, copper, zinc- and manganese superoxide dismutase (CuZnSOD, MnSOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione S-transferase (GST) and glutathione reductase (GR) activities were determined. Cold-induced decrease of CuZnSOD was inhibited with L-NAME, while l-arginine produced the same effect as cold in both supplemented groups. Cold acclimation elevated GSH-Px activity. l-Arginine and L-NAME expressed no effect on GSH-Px in rats kept at room temperature. L-NAME additionally elevated cold-induced GSH-Px activity, l-arginine expressing a similar trend. Cold-induced increase in GST activity was inhibited by L-NAME, while l-arginine inhibited this enzyme in both supplemented groups. Cold acclimation increased GR activity in control and L-NAME-treated group and l-arginine expressed a similar trend. Neither of the treatments affected MnSOD and CAT activities. Cold-induced changes of pancreatic AD were additionally affected by the alterations in l-arginine-NO-producing pathway. Some AD changes in the same direction with l-arginine or L-NAME point to the complexity of nitrogen compounds metabolism and function, accompanied by tissue-specific response.     

Prevalence of scoliosis in school-children from Mostar, Bosnia and Herzegovina

In the school-year 2002/2003 a prospective epidemiological study was performed with the aim of evaluating the prevalence and distribution of scoliosis in the population of schoolchildren from Mostar, Bosnia and Herzegovina. The general check-up of primary-school children covered a total of 2,517 children aged 7-14. The children in which at least one positive symptom of scoliosis was found were directed to undergo orthopedic examination and--if indicated--radiography. Incorrect posture was noted in 33.4% of children, and 11.8% of children were sent to orthopedic specialist examination. The prevalence of structural scoliosis amounted to 3.1%, with the spine curvature threshold being 10 degrees. In eight children (0.32%; 1 boy and 7 girls) a curvature of 20 degrees or more was diagnosed. The most common type of curvature was the thoracal (39%) and the thoraco-lumbar (39%) while 14 children had a double curvature (17.8%). A scoliosis was detected due to here performed check-up in 83.5% of children with scoliosis. No case of serious spine deformity (45 degree or more) was recorded, due to regular general check-ups taking place biannually in this population.     

Pulmonary function in persons who are professionally exposed to formaldehyde fumes

The present study examines long-term effects of occupational exposure to formaldehyde fumes on lung function. Forced spirometry and diffusing lung capacity were measured in 16 health-service professionals (8 medical doctors and 8 laboratory technicians) working at the pathoanatomic laboratory for at least 4 years with daily exposure 8 +/- 1 hours. Control group employed 16 males, which were matched by age and stature to members of the exposed group. Only non-smokers were included in the study. Spirometric parameters in study participants exposed to formaldehyde fumes compared to control group were not significantly different indicating absence of restrictive and/or obstructive deterioration of lung function in exposed group. The only parameter differing in two groups was blood volume of pulmonary capillaries (Vc') which was significantly larger in a group exposed to formaldehyde fumes. The possibility that the hyperemic lung reaction is the consequence of the exposure to formaldehyde fumes should be further explored.     

Dynamics of Cadmium Distribution in the Intercellular Space and Inside Cells in Soybean Roots, Stems and Leaves

Soybean (Glycine max L.) plants grown in nutrient solution were exposed to 1 mM Cd(NO₃)₂ for 24 h. Dynamics of distribution of cadmium among its different forms (water soluble, Ca-exchangeable and complexed) in the intercellular space and the ratio of the intercellular and intracellular cadmium in roots, stems and leaves were studied. In roots, in the beginning of treatment the largest portion of Cd was found in the intercellular space and 1 h later Cd content started to decrease, so that between 13- and 24-h treatment an equilibrium was reached in which about 70 % of Cd was found inside cells. In stems, already after 1-h treatment, the Cd concentrations in the cells and intercellular space were similar, the equilibrium being disturbed after 13 h, so that after 24-h treatment 80 % of Cd was found inside cells. In leaves, up to the 13 h Cd distribution showed fluctuation, after that equilibrium was reached, with 70 % of intracellular Cd. The highest contents of all Cd forms in the intercellular space was observed in roots.     

Artificial neural networks in the modeling and optimization of aspirin extended release tablets with eudragit L 100 as matrix substance

The purpose of the present study was to model the effects of the concentration of Eudragit L 100 and compression pressure as the most important process and formulation variables on the in vitro release profile of aspirin from matrix tables formulated with Eudragit L 100 as matrix substance and to optimize the formulation by artificial neural network. As model formulations, 10 kinds of aspirin matrix tablets were prepared. The amount of Eudragit L 100 and the compression pressure were selected as causal factors. In vitro dissolution time profiles at 4 different sampling times were chosen as responses. A set of release parameters and causal factors were used as tutorial data for the generalized regression neural, network (GRNN) and analyzed using a computer. Observed results of drug release studies indicate that drug release rates vary widely between investigated formulations, with a range of 5 hours to more than 10 hours to complete dissolution. The GRNN model was optimized. The root mean square value for the trained network was 1.12%, which indicated that the optimal GRNN model was reached. Applying the generalized distance function method, the optimal tablet formulation predicted by GRNN was with 5% of Eudragit L 100 and tablet hardness 60N. Calculated difference (f ₁ 2.465) and similarity (f ₂ 85.61) factors indicate that there is no difference between predicted and experimentally observed drug release profiles for the optimal formulation. This work illustrates the potential for an artificial neural network, GRNN, to assist in development of extended release dosage forms.