Genotoxicity of drinking water disinfection by-products (bromoform and chloroform) by using both Allium anaphase-telophase and comet tests

Genotoxic effects of bromoform and chloroform, disinfection by-products of the chlorination of drinking water, were examined by using mitotic index (MI), mitotic phase, chromosome aberrations (CAs) and comet assay on root meristematic cells of Allium cepa. Different concentrations of bromoform (25, 50, 75 and 100 μg/mL) and chloroform (25, 50, 100 and 200 μg/mL) were introduced to onion tuber roots. Distilled water was used as a negative control and methyl methansulfonate (MMS-10 μg/mL) as positive control. All obtained data were subjected to statistical analyses by using SPSS 15.0 for Windows software. For comparison purposes, Duncan multiple range tests by using one-way analysis of variance were employed and p < 0.05 was accepted as significant value. Exposure of both chemicals (except 25 μg/mL applications of bromoform) significantly decreased MI. Bromoform and chloroform (except 25 μg/mL applications) increased total CAs in Allium anaphase-telophase test. A significant increase in DNA damage was also observed at all concentrations of both bromoform and chloroform examined by comet assay. The damages were higher than that of positive control especially at 75–100 μg/mL for bromoform and 100–200 μg/mL for chloroform.     

Production and characterization of Escherichia coli enterohemolysin and its effects on the structure of erythrocyte membranes

Hemolysins are cell-damaging protein toxins produced by pathogenic bacteria, which are usually released into the extracellular medium. Escherichia coli enterohemolysin is an intracellular toxin produced during the log phase of growth, with a maximal intracellular accumulation in the late log phase. In the present study, we have employed electron microscopy and SDS-PAGE to assess the effects of enterohemolysin on erythocyte membranes from different species. The erythrocyte cell damage began immediately after exposure to enterohemolysin with chemically detectable changes in cell membrane permeability, and the formation of surface lesions which increased rapidly in size. This process resulted in complete cell destruction. Ring-shaped structures with a diameter of 10nm were observed by electron microscopy after treatment of horse erythrocyte membranes with enterohemolysin. The ring structures were found clustered and irregularly distributed on the surface of the membranes. Following incubation of the toxin with horse erythrocyte ghosts and detergent-solubilization, the enterohemolysin was isolated from the cytoplasm in its membrane-bound form by sucrose density gradient. SDS-PAGE and silver staining of deoxycholate-solubilized target membranes revealed heterogeneous forms of the toxin. By using SDS-PAGE and gel filtration, the molecular weight of the toxin was estimated to be 35 kDa. With respect to species specificity, horse erythrocytes showed the highest sensitivity to the enterohemolysin, followed by human and guinea pig erythrocytes. The hemolytic sensitivity correlated with the toxin binding capacity of erythrocyte membranes of different animal species. The degree of hemolysis was unaffected by temperature in the range of 4 degrees C-37 degrees C and was optimal at pH 9.0. In contrast to pore-forming cytolysins, the hemolytic activity of enterohemolysin was enhanced continuously in the presence of increasing concentrations of dextran 4 and dextran 8 within the range of 5 to 30 mM. Trypsin sensitivity of membrane-bound enterohemolysin indicates that the cell surface is the most likely target site for this toxin. Additionally, the fact that proteinase and phosphatase inhibitors failed to inhibit lysis suggests that enterohemolysin alters and disrupts cell membranes by a detergent-like mechanism.     

New suspension culture system (Mini-AR).

A new suspension culture system (Mini-AR) based on the Stoke's drag law for suspended particles is described. This apparatus can be utilized for the maintenance of mammalian kidney cells in both short- and long-term cultures.     

The Effects of Flaxseed Oil on Cadmium-Induced Oxidative Stress in Rats

In the present study, the effects of flaxseed oil on the oxidant–antioxidant system in cadmium intoxication were investigated in rats. Forty-eight male Wistar albino rats were divided into four equal groups (group 1). No treatment was applied to the control group. On the other hand, groups 2, 3, and 4 were administered with 0.1 ml/rat/day (～500 mg/kg bw) flaxseed oil by gavage into the stomach, 50 ppm of cadmium (～4 mg/kg bw) in ad libitum drinking water, and 0.1 ml/rat/day flaxseed oil plus 50 ppm of cadmium, respectively, for 30 days. At the end of the study, malondialdehyde and nitric oxide levels and catalase, superoxide dismutase, and glutathione peroxidase activities were measured in blood and tissue (liver, lung, kidney, brain, heart, and testes) samples. While malondialdehyde and nitric oxide levels increased in the group given cadmium compared to the control group; in the meantime, there were some significant changes in antioxidant enzyme activities. These changes were observed, the trends of decrease or increase compared to the control group. There were positive changes in parameters of the group given with flaxseed oil plus cadmium compared to the group receiving cadmium alone, in other words, values were seen coming close to control group. As a result, cadmium exposure caused oxidative damage to erythrocytes and organs at varying rates, while flaxseed oil reduced the severity of cadmium-induced lipid peroxidation. Therefore, it was concluded that flaxseed oil can be used among compounds as a therapeutic agent or food additive for prophylaxis in cadmium intoxication.     

Effect of drought stress implemented at pre- or post-anthesis stage on some physiological parameters as screening criteria in chickpea cultivars

Drought is one of the most important factors limiting chickpea production in arid and semi-arid regions. There is little information regarding genotypic variation for drought tolerance in chickpea cultivars. Screening for drought tolerance is very important. It is essential to identify the physiological mechanisms of drought tolerance to complete conventional breeding program. Glasshouse experiment was carried out to study the genotypic variation among 11 chickpea (Cicer arietinum L.) cultivars. Plants were grown either under optimum conditions or drought stress was implemented at pre-or post-anthesis stages. The drought susceptibility index (DSI) was used as the measure of drought tolerance. Relationships between DSI and excised-leaf water loss (RWL), relative water content (RWC), membrane permeability, ascorbic acid, proline, and chlorophyll contents, lipid peroxidation, and hydrogen peroxide concentrations were determined in order to find out whether these physiological parameters could be used as the genotypic selection criteria for drought tolerance. The results of this study indicated that there was a wide variation in tolerance to drought stress among the chickpea cultivars, which could be exploited in breeding new chickpea cultivars with high drought tolerance. The results also demonstrated that drought-tolerant cultivars had a higher RWC, ascorbic acid and proline concentrations, but lower RWL and membrane permeability in comparison to drought-sensitive cultivars. The significant and a well defined relationships between DSI and RWC, RWL, ascorbic acid, proline, and membrane permeability were found. It was concluded that these parameters could be instrumental in predicting the drought tolerance of chickpea cultivars. This text was submitted by the authors in English.     

Diagnosis of hepatocellular carcinoma by fine needle aspiration cytology. Cellular features

OBJECTIVE: To evaluate the importance of fine needle aspiration cytology (FNAC) in the diagnosis of hepatocellular carcinoma (HCC). STUDY DESIGN: We analyzed 17 cytologic and 5 architectural features in a series of 320 FNACs from HCC and compared them with 73 FNACs from benign lesions and with 705 FNACs from metastatic carcinomas. One thousand ninety-eight patients who were diagnosed by liver FNAC between December 1988 and July 1998 and had adequate follow-up were included in the study. The specimens were evaluated according to the presence or absence of the cytologic features and cellular arrangement. A stepwise logistic regression analysis was performed on the data to determine the variables predictive of HCC. RESULTS: Multinucleated tumor giant cells, cytoplasmic hyaline and central sinusoidal pattern were selected as the 3 most predictive parameters for differentiated reactive hepatocytes from HCC (P < .0001), while bile, centrally located nucleus in an atypical cell and intranuclear inclusion were selected as the 3 most predictive parameters for differentiated metastatic carcinoma from HCC (P < .0001-< .001) by stepwise logistic regression analysis. CONCLUSION: In the 1,098 patients suspected of having hepatic malignancy, a correct diagnosis was made by a combination of the above features. The sensitivity of this procedure for hepatic malignancy was 99.5%, and the specificity was 100%.     

Balamuthia mandrillaris, free-living ameba and opportunistic agent of encephalitis, is a potential host for Legionella pneumophila bacteria

Balamuthia mandrillaris is a free-living ameba and an opportunistic agent of granulomatous encephalitis in humans and other mammalian species. Other free-living amebas, such as Acanthamoeba and Hartmannella, can provide a niche for intracellular survival of bacteria, including the causative agent of Legionnaires' disease, Legionella pneumophila. Infection of amebas by L. pneumophila enhances the bacterial infectivity for mammalian cells and lung tissues. Likewise, the pathogenicity of amebas may be enhanced when they host bacteria. So far, the colonization of B. mandrillaris by bacteria has not been convincingly shown. In this study, we investigated whether this ameba could host L. pneumophila bacteria. Our experiments showed that L. pneumophila could initiate uptake by B. mandrillaris and could replicate within the ameba about 4 to 5 log cycles from 24 to 72 h after infection. On the other hand, a dotA mutant, known to be unable to propagate in Acanthamoeba castellanii, also did not replicate within B. mandrillaris. Approaching completion of the intracellular cycle, L. pneumophila wild-type bacteria were able to destroy their ameboid hosts. Observations by light microscopy paralleled our quantitative data and revealed the rounding, collapse, clumping, and complete destruction of the infected amebas. Electron microscopic studies unveiled the replication of the bacteria in a compartment surrounded by a structure resembling rough endoplasmic reticulum. The course of intracellular infection, the degree of bacterial multiplication, and the ultrastructural features of a L. pneumophila-infected B. mandrillaris ameba resembled those described for other amebas hosting Legionella bacteria. We hence speculate that B. mandrillaris might serve as a host for bacteria in its natural environment.     

IL-6 is required for protective immune responses against early filarial infection

IL-6 has a wide range of biological activities that includes anti- and pro-inflammatory aspects. In this study, we investigated the role of IL-6 in immune responses to the rodent filarial nematode Litomosoides sigmodontis, a model for human filarial infections. IL-6^?/? mice had a significantly increased worm burden after natural infection compared with wild type controls at early time points p.i. Given that the worm burden in IL-6^?/? mice was already increased at the time point the infective larvae reached the pleural cavity, immune responses that may facilitate the migration from the site of infection (skin) via the lymphatics to the pleural cavity were analysed. Increased vascular permeability may facilitate larval migration, but blocking of histamine receptors had no effect on worm burden and vascular permeability was similar between IL-6^?/? mice and wild type controls. In contrast, blocking mast cell degranulation reduced the worm burden in IL-6^?/? mice partially, suggesting that release of mast cell-derived mediators improves larval migration to some degree. Protective immune responses within the skin were involved, as bypassing the skin barrier by inoculating infective L3s subcutaneously resulted in a comparable worm recovery in both mouse strains. Analysis of the cellular composition by flow cytometry and PCR array in the skin after exposure to filarial extract or L3s, respectively, indicate that the absence of IL-6 results in a delayed recruitment of neutrophils and macrophages to the site of initial infection. These results demonstrate that IL-6 is essentially involved in protective immune responses within the skin that impair migration of infective L3s.