Acanthamoeba castellanii: Cellular changes induced by chlorination

Chlorination is a well-known disinfection method, used in water treatment to inactivate various microorganisms, it induces numerous cellular changes. Even though Acanthamoebae are frequently found in water, the cellular changes induced in Acanthamoebae have not been described in the literature. Acanthamoebae are pathogenic amoebae and may provide a reservoir for pathogenic bacteria such as Legionellapneumophila; it is consequently important to understand the response of this amoeba to chlorination, and our study was indeed aimed at examining cellular changes in Acanthamoebae following chlorination. Acanthamoeba trophozoites were treated at various chlorine concentrations (1-5 mg/L). A 3-log reduction in Acanthamoebae population was achieved with 5 mg/L of free chlorine. Confocal microscopy and flow cytometry experiments indicated that chlorination induced cell permeabilization, size reduction and likely intracellular thiol concentration. Our data show that among the non-cultivable cells some remained impermeabilized (negative staining with propidium iodide), thereby suggesting that these cells might remained viable. A similar state is described in other microorganisms as a VBNC (viable but not cultivable) state. Electron microscopy observations illustrate drastic morphological changes: the pseudopods disappeared and subcellular components, such as mitochondrion, were pronouncedly affected. In conclusion, depending on the concentration used, chlorination leads to many cellular effects on Acanthamoeba that could well arise in cell inactivation.     

Deletions induced by gamma rays in the genome of Escherichia coli.

An Escherichia coli lysogen was constructed with a lambda phage bearing a lacZ gene surrounded by about 100 x 10(3) base-pairs of dispensable DNA. The lacZ mutants induced by gamma rays in this lysogen were more than 10% large deletions, ranging in size from 0.6 x 10(-3) to 70 x 10(3) base-pairs. These deletions were centered, not on lacZ, but on a ColE1 origin of DNA replication located 1.2 x 10(3) bases downstream from lacZ. This suggested that this origin of replication was involved in the process by which the deletions were formed. In agreement with this hypothesis, a lysogen of the same phage without the ColE1 origin showed a very much lower percentage of radiation-induced deletions, as did a second lysogen of a lambda phage without any known plasmid origin of replication. Indirect evidence is presented for radiation-induced deletions centered on the lambda origin of DNA replication in a lysogen. It is suggested that high percentages of large deletions may occur among radiation-induced mutations in mammalian cells because deletions centered on some of the thousands of origins of replication in these genomes do not kill the cells.     

Alterations in Solanum tuberosum polyphenol oxidase activity induced by gamma irradiation

On gamma irradiation of potato tubers at sprout-inhibiting dose (10 krad) the cresolase activity showed a 45% increase while catecholase was reduced by 25%. This reduced the ratio of catecholase to cresolase from 11–12 in unirradiated to 5–6 in irradiated potatoes. Chlorogenic acid oxidation was enhanced by about 25% on irradiation. The increase in the oxidation of p-cresol corresponded with the production of diphenolic compounds. The process of activation of cresolase was slow, reversible and oxygen dependent. A comparative study of the isoenzyme pattern suggested that this activation was due to conformational change, rather than synthesis of new protein.     

Complex chromosome exchanges induced by gamma rays in human lymphocytes: an mFISH study

Loucas, B. D. and Cornforth, M. N. Complex Chromosome Exchanges Induced by Gamma Rays in Human Lymphocytes: An mFISH Study. Radiat. Res. 155, 660-671 (2001). Combinatorial multi-fluor fluorescence in situ hybridization (mFISH) allows the simultaneous painting of each pair of homologous chromosomes, thereby eliminating many of the difficulties previously associated with the analysis of complex rearrangements. We employed mFISH to visualize exchanges in human lymphocytes and found significant frequencies of these aberrations after gamma-ray doses of 2 and 4 Gy. At 4 Gy, roughly half of the cells contained at least one complex exchange that required anywhere from 3 to 11 initial chromosome breaks. At this dose, more than 40% of gross cytogenetic damage, as measured by the total number of exchange breakpoints, was complex in origin. Both simple and complex exchanges were found to have nonlinear dose responses, although the latter showed significantly more upward curvature. In many cases, it could be deduced that the initial breaks leading to a particular complex exchange were proximate, meaning that the resulting broken chromosome ends all must have been capable of interacting freely during the exchange process. For other complex exchanges, the rearrangement could just as well have resulted from two or more simpler exchanges that occurred sequentially. The results demonstrate the utility of mFISH in visualizing intricacies of the exchange process, but also highlight the various sources of ambiguity concerning cytogenetic analysis that remain despite the power of this approach.     

Reciprocal adaptive response of human peripheral lymphocytes induced by bleomycine or gamma rays.

The adaptive response and reciprocal adaptive response induced in vitro by exposure to low doses of gamma rays (0.05 Gy) or bleomycin (0.05 microg/ml) in human peripheral blood lymphocytes were assessed by the frequency of chromosome aberrations. Gamma rays (1.5 Gy) or bleomycin (1.5 microg/ml) were used as the challenge doses. In the experiments, blood samples from 5 healthy donors were investigated. It has been found that low doses of bleomycin and gamma rays induced a reciprocal adaptive response to high doses of gamma rays or bleomycin. Moreover, the results confirmed that the adaptive response did not correlate with the radiosensitivity of the peripheral blood lymphocytes.     

The repair of DNA damage induced in human lymphocytes by gamma rays and fast neurons

When Go human lymphocytes are exposed either to gamma-rays or to d(50)-Be neutrons and then immediately incubated in presence of cytosine arabinoside, the frequency of chromosomal aberrations which is normally observed after radiation exposure only is sharply increased. This enhancement of the aberrations, particularly the dicentrics, is, however, less marked when cytosine arabinoside is administered at longer intervals of time after irradiation. For gamma-rays, the treatment with cytosine arabinoside has no effect on the dicentrics yield when given 5 h after irradiation, indicating that the repair is completed within the 5 h after irradiation and that the lesions are not anymore available to produce exchange aberrations. For d(50)-Be neutrons, the time of repair takes approximately 5 h after a dose of 2.0 Gy, whereas it appears to be shorter (3 h) after a dose of 0.5 Gy.     

Rapid acclimatization to low temperature in the Queensland fruit fly, Dacus tryoni

Dacus tryoni, which overwinters in conditions of wide diurnal fluctuations in temperature, is capable of rapid acclimatization with respect to thresholds for torpor and flight but ability for metabolic compensation is apparently lacking. The rate of reacclimatization on abrupt transfer to lower temperatures is quicker the less the temperature drops and extrapolation of this relationship suggests a time of the order of 1 min for a drop as small as 1°C. This explains why continuous reacclimatization appears to take place during gradual cooling, even when this is as fast as 1°C/min. The insect can rapidly produce its maximum response to extensive drops of temperature as long as the latter falls gradually at a rate no faster than 1°C/min. Acclimatization in nature is therefore ‘immediate’. Reacclimatization to higher temperatures (deacclimatization to cold) depends upon the temperature that the insects are raised to and thus is slower the smaller the rise and understandably is not accomplished more rapidly by a gradual transition.     

Potential ultrastructural changes in rat epididymal cell types induced by Boswellia papyrifera and Boswellia carterii incense

Boswellia papyrifera and Boswellia carterii, known as Arabian incense, diffuses smoke, contaminating the air, which adversely affects human health. Therefore, this study was designed to ascertain the effect of these plants on histopathological and ultrastructure changes in cauda epididymis of Albino rats. Animals were exposed to 4 g/kg body weight of B. papyrifera and B. carterii daily for 120 days along with suitable controls. Our study indicates a significant reduction in epithelial heights. Cells showed signs of degeneration. The ultrastructural study revealed that the cauda epididymis was affected, including its cell types. Furthermore, a decrease in the size of mitochondria, Golgi complex, and both ERs was observed. In all treated groups, plasma fructose decreased considerably, indicating the sign of reduced energy, vital for motility and other sperm functions. The results of this study suggest that these plants systematically affect cauda epididymal cell types and its lumen through its potential toxicity.     

Metamorphic changes of wild type and mutant Drosophila tissues induced by 20-hydroxy ecdysone in vitro

Wild type (Oregon R) and non-pupariating as well as late-pupariating mutant larval tissues were cultured in vitro up to 5 weeks with and without 20-hydroxy ecdysone (1 μg/ml). The following responses were elicited by the hormone: in the case of wild type tissues detachment of the larval epidermis and muscles from the cuticle; puparial tanning and sclerotization of the larval cuticle; dissociation of the fat body into single cells; inhibition of the movement of the hind intestine. Most of these responses developed within 1 week of culturing. Of the 4 mutants tested, 3 behaved like the wild type. In cultures of ?(1)npr-1, however, puparial tanning, disc evagination, and inhibition of the movement of the hind intestine was abnormally weak and the dissociation of fat body was not observed at all. Detachment of the epidermis and muscles as well as formation of the pupal cuticle by disc tissue occurred normally. The results are discussed with respect to the ecdysteroid-induced metamorphosis of the tissues and the autonomy of mutant gene action.     

Ultrastructural and hormonal changes in rat cauda epididymal spermatozoa induced by Boswellia papyrifera and Boswellia carterii

Boswellia papyrifera and Boswellia carterii diffuses smoke polluting air that adversely affects indoor environment that certainly harm human health. Therefore, this study aims at ascertaining the effect of these plants on gonadal hormones and molecular changes in rat spermatozoa. The animals were exposed to 4 g/kg body weight of B. papyrifera and B. carterii daily for 120 days along with suitable controls. Significant decreases in FSH, LH and testosterone levels were evidenced, along with a reduction of protein, sialic acid, and carnitine levels. In sperm physiology, sperm count, motility, speed decrease, whereas sperm anomalies increase. TEM observation indicates morphological changes in plasma and acrosomal membranes, cytoplasmic droplet in the tail region, vacuolated, and disorganization of the mitochondrial sheath. These findings demonstrate that B. papyrifera and B. carterii smoke affects the process of sperm formation and maturation, which indicates the detrimental effects of these plants on the reproductive system.     

Intragenic mitotic recombination induced by ultraviolet and gamma rays in radiosensitive mutants of Saccharomyces cerevisiae yeasts

The effect of UV- and gamma-irradiation on the survival and intragenic mitotic recombination (gene conversion) of 5 radiosensitive mutants was studied in comparison with the wild type. The level of spontaneous conversion was similar for RAD, rad2 and rad15, mutations xrs2 and xrs4 increasing and rad54 significantly decreasing it. The frequency of conversion induced by UV-light was greater in rad2, rad15 and xrs2 mutants and lower in xrs4, as compared to RAD. Gamma-irradiation caused induction of gene conversion with an equal frequency in RAD, rad2, rad15. Xrs2 and xrs4 mutations slightly decreased gamma-induced conversion. In rad54 mutant, UV-and gamma-induced conversion was practically absent. In the wild type yeast, a diploid strain is more resistant than a haploid, whereas in rad54 a diploid strain has the same or an increased sensitivity, as compared to a haploid strain (the "inverse ploidy effect"). This effect and also the block of induced mitotic recombination caused by rad54 indicate the presence in the yeast Saccharomyces cerevisiae of repair pathways of UV- and gamma-induced damages acting in diploid cells and realised by recombination. The data obtained as a result of many years' investigation of genetic effects in radiosensitive mutants of yeast are summarised and considered.     

Morphological changes of human neuroblastoma cells induced by human gamma interferon

The treatment of GOTO cells, originated from human neuroblastoma, with recombinant human interferon-gamma (rHuIFN-gamma) induced the morphological changes: the extension and bifurcation of neurites and the multinucleated giant cell formation. The treatment of KP-N-RT cells, originated from human neuroblastoma, with rHuIFN-gamma also induced the similar morphological changes. The treatment of these cells with natural HuIFN-gamma also induced the same morphological changes, but those with recombinant human leukocyte interferon (rHuIFN-alpha A), recombinant human fibroblast interferon (rHuIFN-beta) and recombinant murine interferon-gamma (rMuIFN-gamma) did not induce it. The rHuIFN-gamma and the rHuIFN-beta inhibited more strongly the growth of GOTO and KP-N-RT cells than the rHuIFN-alpha A. This suggests that the morphological changes of these neuroblastoma cells are not simply due to the cell growth inhibition, but due to the property which only the rHuIFN-gamma possesses.     

Behavioural responses to visual stimuli by the snail Littorina irrorata

The visual capabilities of gastropod molluses and most other invertebrates possessing structurally simple eyes are poorly known. We studied vision in untrained marsh periwinkles (Littorina irrorata) in the laboratory, using oriented movements toward test shapes as the response measure. This intertidal species is active when exposed at low tide, both during the day and at night, and it travels vertically on plant stems with a tidal rhythm. In detection tests, the estimated response threshold for a single vertical bar was 0.9°, while the response threshold for an equal-size horizontal bar was 2.4° or 3.7°, depending on bar position. Snails detected a 5°-wide bar in 4.3 1x of light and a 40°-wide square having about 95% reflectance (‘off-white’) on a white (100% nominal reflectance) background in 2800 1x. Discrimination tests revealed a strong preference for vertical bars over both diagonal and horizontal bars of the same width, but no preferences in several other situations. Various factors suggest that L. irrorata may see better than most other gastropods.     

Elevation of glutathione induced by low-dose gamma rays and its involvement in increased natural killer activity

We examined the relationship between the induction of an increase in the level of glutathione and the elevation of natural killer (NK) activity in mouse splenocytes by a low dose of gamma rays. The glutathione levels in mouse splenocytes increased significantly between 2 h and 6 h after whole-body gamma irradiation at 0.5 Gy, peaked at 4 h, and then decreased almost to the level before irradiation by 12 h postirradiation. A significant enhancement of NK activity was found in the splenocytes obtained from whole-body-irradiated mice between 4 and 6 h postirradiation. Reduced glutathione (GSH) added exogenously to splenocytes obtained from normal mice enhanced both the total cellular glutathione content and the NK activity in a dose-dependent manner. Other precursors of de novo GSH synthesis, such as cysteine, N-acetylcysteine and oxidized glutathione, also increased the activity. These enhancements were completely blocked by buthionine sulfoximine, an inhibitor of de novo GSH synthesis. We conclude that the induction of endogenous glutathione in living cells immediately after low-dose gamma irradiation is at least partially responsible for the appearance of enhanced NK activity.     

Measurement of the initial levels of DNA damage in human lymphocytes induced by 29 kV X rays (mammography X rays) relative to 220 kV X rays and gamma rays

Experiments using the alkaline comet assay, which measures all single-strand breaks regardless of their origin, were performed to evaluate the biological effectiveness of photons with different energies in causing these breaks. The aim was to measure human lymphocytes directly for DNA damage and subsequent repair kinetics induced by mammography 29 kV X rays relative to 220 kV X rays, 137Cs gamma rays and 60Co gamma rays. The level of DNA damage, predominantly due to single-strand breaks, was computed as the Olive tail moment or percentage DNA in the tail for different air kerma doses (0.5, 0.75, 1, 1.5, 2 and 3 Gy). Fifty cells were analyzed per slide with a semiautomatic imaging system. Data from five independent experiments were transformed to natural logarithms and fitted using a multiple linear regression analysis. Irradiations with the different photon energies were performed simultaneously for each experiment to minimize interexperimental variation. Blood from only one male and one female was used. The interexperimental variation and the influence of donor gender were negligible. In addition, repair kinetics and residual DNA damage after exposure to a dose of 3 Gy were evaluated in three independent experiments for different repair times (10, 20, 30 and 60 min). Data for the fraction of remaining damage were fitted to the simple function F(d) = A/(t + A), where F(d) is the fraction of remaining damage, t is the time allowed for repair, and A (the only fit parameter) is the repair half-time. It was found that the comet assay data did not indicate any difference in the initial radiation damage produced by 29 kV X rays relative to the reference radiation types, 220 kV X rays and the gamma rays of 137Cs and 60Co, either for the total dose range or in the low-dose range. These results are, with some restrictions, consistent with physical examinations and predictions concerning, for example, the assessment of the possible difference in effectiveness in causing strand breaks between mammography X rays and conventional (150-250 kV) X rays, indicating that differences in biological effects must arise through downstream processing of the damage.