Mutation of human lymphoblasts by methylnitrosourea

The lag in phenotype expression of methylnitrosourea(MNU)-induced mutation to 6-thioguanine (6TG) resistance has been studied in a diploid human lymphoblastoid cell line. We find that a considerable period (8–12 days) elapses before new mutants appear in treated cultures; after 2 weeks, however, a stable maximum fraction is attained, as would be expected for a genetic mutation. We present preliminary data linking this phenotypic lag to the slow degradation rate of hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and to an apparent requirement for very low (<0.2% normal) cellular HGPRT content in order for cells to be resistant to 10 μg 6TG/ml. A series of reconstruction experiments are presented, the results of which support the conclusion that selective pressures in the assay procedure do not bias the quantitative estimates of induced mutant fraction.     

Production of 1,2-diacylglycerol in human erythrocyte membranes exposed to low concentrations of calcium ions

A specific increase in the membrane content of 1,2-diacylglycerol occurred when erythorcytes were lysed at 20 °C in media which did not include a chelator of Ca²⁺ and also when Ca²⁺ was added to haemoglobin-free erythrocyte ghosts which had been prepared in the presence of ethyleneglycol-bis-(β-aminoethylether)-N,N′-tetraacetic acid (EGTA). The maximum increase was about 20-fold. The production of 1,2-diacylglycerol appeared to be caused by an endogenous membrane-bound phospholipase C which was half-maximally activated at less than 1 μM Ca²⁺ and which had access to only about 0.6–0.8% of the cells' glycerolipids. This activity was optimal at pH 7.0–7.2 in the presence of 0.1 mM Ca²⁺; under these conditions diacylglycerol production was complete within 5–10 min. Enzyme activity was markedly decreased at low temperatures, and was abolished by heating at 100 °C for 1 min.     

Differentiation of activity of a superoxide dismutase inhibitor in human cells exposed to radiation, chemical mutagens and radioadaptive response

The superoxide dismutase (SOD) inhibitor, TRIEN, which enhanced the formation of gamma-induced DNA breaks in cells of healthy donors and patients with Marfan syndrome and Bloom syndrome (repair-defective hereditary diseases), had virtually no effect on the formation of radioadaptive response (RAR) in these systems. Similar results were obtained in studies on cell survival: TRIEN facilitated mortality in cells irradiated with gamma-rays but did not affect RAR formation. TRIEN also increased the deleterious effect of CdCl2, which indicates that SOD apparently plays a certain role in cell defence against this mutagen.     

Mineral status and mechanical properties of cancellous bone exposed to hydrogen peroxide for various time periods

Processed cancellous bone has been regarded as one alternative for the treatment of bone defects. In order to avoid immunogenic effects and preserve the natural properties of the bone, the optimal processing method should be determined. To observe the influence of hydrogen peroxide on the mineral status and mechanical properties of cancellous bone for various time periods and find the optimal processing time. Cancellous bone granules from bovine femur condyles were treated with 30% hydrogen dioxide for 0, 12, 24, 36, 48, 60 and 72 h separately. The microstructure and mineral content of the granules were evaluated by ash analysis, Micro-CT, scanning electron micrograph and energy dispersive X-ray. The biomechanical properties were analyzed by applying cranial-caudal compression in a materials testing machine. With increasing exposure to hydrogen peroxide, the BMD and BMC of granules gradually decreased, and the Ca/P molar ratios clearly increased (P < 0.05). Meanwhile, the mineral content of the granules increased from 48.5 ± 1.3 to 79.5 ± 2.1%. Substantial decreases in the strength of the granules were observed, and after 48 h severe decreases were noted. The decrease in strength was also evident after normalizing the parameters to the cross-sectional area. Granules of bovine cancellous bone matrix should be processed by hydrogen peroxide for 12 to 36 h to fulfill the basic requirements of a bone tissue engineering scaffold. These granules could potentially be useful during orthopedic operations.     

Sensitivity of Drosophila melanogaster to low concentrations of gaseous mutagens. II. Chronic exposures.

Sex-linked recessive lethal mutations were induced in Drosophila melanogaster males by gaseous 1,2-dibromoethane at concentrations ranging from 0.2 to 2 parts per million. Significant numbers of mutations could be induced at all these concentrations. Pronounced germ-cell sensitivity differences were observed. For low exposures, spermatids and spermatocytes were about 10--20 times more sensitive than spermatozoa. The dose-effect relation was linear below 60 ppm . h for the 3 cell types. At higher exposures, sterility prevented mutation detection in spermatocytes and in spermatogonia. The lowest effective exposure for spermatozoa was 18 ppm . h (0.25 ppm for 72 h). In spermatids, the lowest exposure tested, 2.3 ppm . h (0.2 ppm for 11 h) induced 4 times the spontaneous mutation rate. Therefore, using prolonged exposure periods one may be able to detect concentrations in the range of parts per billion. Thus, Drosophila appears suitable as a system for detecting very low concentrations of gaseous mutagens in industrial, agricultural and environmental atmospheres.     

Human lymphocytes exposed to low doses of ionizing radiations become refractory to high doses of radiation as well as to chemical mutagens that induce double-strand breaks in DNA

Human lymphocytes exposed to low doses of ionizing radiation from incorporated tritiated thymidine or from X-rays become less susceptible to the induction of chromatid breaks by high doses of X-rays. This response can be induced by 0.01 Gy (1 rad) of X-rays, and has been attributed to the induction of a repair mechanism that causes the restitution of X-ray-induced chromosome breaks. Because the major lesions responsible for the induction of chromosome breakage are double-strand breaks in DNA, attempts have been made to see if the repair mechanism can affect various types of clastogenic lesions induced in DNA by chemical mutagens and carcinogens. When cells exposed to 0.01 Gy of X-rays or to low doses of tritiated thymidine were subsequently challenged with high doses of tritiated thymidine or bleomycin, which can induce double-strand breaks in DNA, or mitomycin C, which can induce cross-links in DNA, approximately half as many chromatid breaks were induced as expected. When, on the other hand, the cells were challenged with the alkylating agent methyl methanesulfonate (MMS), which can produce single-strand breaks in DNA, approximately twice as much damage was found as was induced by MMS alone. The results indicate that prior exposure to 0.01 Gy of X-rays reduces the number of chromosome breaks induced by double-strand breaks, and perhaps even by cross-links, in DNA, but has the opposite effect on breaks induced by the alkylating agent MMS. The results also show that the induced repair mechanism is different from that observed in the adaptive response that follows exposure to low doses of alkylating agents.     

Stressful effects of chemical toxins at low concentrations

Effects of three chemical compounds: ammonia, diethyl ether, and acetic acid, known as common environmental contaminants in technogenic accidents, were investigated in vivo and in vitro in low concentrations. When added in cultivation media, each of the chemicals has affected peritoneal macrophages and spleen lymphocytes isolated from male NMRI mice and led to a rise in the production of several cytokines, particularly the tumor necrosis factor-α and interferon-γ, as well as the expression of the inducible form of heat shock proteins (HSP72 and HSP90-α) and in the activation of signal cascades NF-κB and SAPK/JNK. The increase of the nitric oxide (NO) production in macrophages has been observed only when ammonia was added in cultivation media. Also, low concentrations of all compounds investigated led to the activation of the expression of receptor protein TLR4. When mice were exposed to airborne toxic contaminants in a hermetically sealed experimental chamber, an increase in the concentrations of cytokines, heat shock proteins, and signal proteins in immune cells was also observed in response to low concentrations of all chemicals investigated. Similarly to in vitro experiments, the NO production was augmented only in the presence of the airborne ammonia. The results indicate the environmental hazard of chemical contaminants even in rather low concentrations, which nevertheless lead to the stress response.     

Induction of HLA mutations by chemical mutagens in human lymphoid cells

We studied the effects of mutagens on the frequency of HLA loss variants in the diploid human lymphoid cell line T5-1, as well as the stability of mutagen-induced variants and the extent of the genetic lesions induced. Mutagens used were of different types, and included ethyl methanesulfonate (EMS), ICR-191 (ICR), and mitomycin C. Variant frequency was determined seven days after a 24-hour exposure to mutagen, by which time the cloning efficiency of exposed cultures, which was reduced following mutagenesis, had returned to normal. ICR and EMS induced dose-related increases in variant frequency of greater than two orders of magnitude at the highest concentrations tested. Mitomycin C increased variant frequency by tenfold at the one concentration tested. Seventeen of 18 induced variants showed persistence of the variant phenotype during prolonged culture following isolation. Genetic lesions induced by ICR and EMS did not extend as far as the distance between theHLA-A and -B loci (0.8 map units) in any of 21 clones tested. These data suggest a mutational origin for most mutagen-induced HLA variants of diploid cells (except for some induced by mitomycin C). Mutagenesis and mutational analysis are promising probes for studies of theHLA region.Abbreviations used in this paper are as follows MHC major histocompatibility complex - EMS ethyl methanesulfonate - ICR ICR-191 - C complement     

Synthesis and activity of nitrogenase in Klebsiella pneumoniae exposed to low concentrations of oxygen

Effects of very low concentrations of dissolved O2 on nitrogenase activity in Klebsiella pneumoniae were studied in a stirred chamber system which enabled simultaneous measurements of steady-state O2 concentrations, O2 consumption and C2H2 reduction. A strain carrying a chromosomal nifH::lac fusion as well as the Nif+ plasmid pRD1, expressed nitrogenase activity with 80 nM-O2, a concentration known to inhibit nifH::lac expression by about 50% Thus nitrogenase activity in vivo was no more sensitive to O2 than expression of nifH::lac. When compared with anaerobic treatments, dissolved O2 near 30 nM apparently stimulated nitrogenase derepression and enhanced the activity of nitrogenase synthesized anaerobically. Thus, in this organism, N2 fixation occurs in microaerobic as well as anaerobic conditions.     

Metabolic evidence for stelar anoxia in maize roots exposed to low o(2) concentrations

This investigation presents metabolic evidence to show that in 4- to 5-day-old roots of maize (Zea mays hybrid GH 5010) exposed to low external O₂ concentrations, the stele receives inadequate O₂ for oxidative phosphorylation, while the cortex continues to respire even when the external solution is at zero O₂ and the roots rely solely on aerenchyma for O₂ transport. Oxygen uptake rates (micromoles per cubic centimeter per hour) declined at higher external O₂ concentrations in excised segments from whole roots than from the isolated cortex; critical O₂ pressures for respiration were greater than 0.26 moles per cubic meter O₂ (aerated solution) for the whole root and only 0.075 moles per cubic meter O₂ for the cortex. For plants with their shoots excised and the cut stem in air, ethanol concentrations (moles per cubic meter) in roots exposed to 0.06 moles per cubic meter O₂ were 3.3 times higher in the stele than in the cortex, whereas this ethanol gradient across the root was not evident in roots exposed to 0 moles per cubic meter O₂. Alanine concentrations (moles per cubic meter) in the stele of roots exposed to 0.13 and 0.09 moles per cubic meter O₂ increased by 26 and 44%, respectively, above the levels found for aerated roots, whereas alanine in the cortex was unchanged; the increase in stelar alanine concentration was not accompanied by changes in the concentration of free amino acids other than alanine. For plants with their shoots intact, alcohol dehydrogenase and pyruvate decarboxylase activities (micromoles per gram protein per minute) in roots exposed to 0.13 moles per cubic meter O₂ increased in the stele by 40 to 50% over the activity in aerated roots, whereas there was no appreciable increase in alcohol dehydrogenase and pyruvate decarboxylase activity in the cortex of these roots. More convincingly, for roots receiving O₂ solely from the shoots via the aerenchyma, pyruvate decarboxylase in the cortex was in an “inactive” state, whereas pyruvate decarboxylase in the stele was in an “active” state. These results suggest that for roots in O₂-free solutions, the aerenchyma provides adequate O₂ for respiration in the cortex but not in the stele, and this was supported by a change in pyruvate decarboxylase in the cortex to an active state when the O₂ supply to the roots via the aerenchyma was blocked.     

Morphological changes in Escherichia coli cells exposed to low or high concentrations of hydrogen peroxide

Escherichia coli cells challenged with low or high concentrations of hydrogen peroxide are killed via two different mechanisms and respond with morphological changes which are also dependent on the extracellular concentration of the oxidant. Treatment with low concentrations (less than 2.5 mM) of H2O2 is followed by an extensive cell filamentation which is dependent on the level of H2O2 or the time of exposure. In particular, addition of 1.75 mM H2O2 results in a growth lag of approximately 90 min followed by partial increase in optical density, which was mainly due to the onset of the filamentous response. In fact, microscopic analysis of the samples obtained from cultures incubated with the oxidant for various time intervals has revealed that this change in morphology becomes apparent after 90 min of exposure to H2O2 and that the length of the filaments gradually increases following longer time intervals. Analysis of the ability of these cells to form colonies has indicated a loss in viability in the first 90 min of exposure followed by a gradual recovery in the number of cells capable of forming colonies. Measurement of lactate dehydrogenase in culture medium (as a marker for membrane damage) has revealed that a small amount of this enzyme was released from the cells at early times (less than 150 min) but not after longer incubation periods (300 min). Cells exposed to high concentrations of H2O2 (greater than 10 mM) do not filament and their loss of viability is associated with a marked reduction in cell volume. In fact, treatment with 17.5 mM H2O2 resulted in a time-dependent decrease of the optical density, clonogenicity, and cellular volume. In addition, these effects were paralleled by a significant release in the culture medium of lactate dehydrogenase thus suggesting that the reduced cell volume may be dependent on membrane damage followed by loss of intracellular material. This hypothesis is supported by preliminary results obtained in electron microscopy studies. In conclusion, this study further demonstrates that the response of E. coli to hydrogen peroxide is highly dependent on the concentration of H2O2 and further stresses the point that low or high concentrations of the oxidant result in the production of different species leading to cell death via two different mechanisms and/or capable of specifically affecting the cell shape.     

Optimization of protective properties of interferons in human cells exposed to mutagens estimated by the DNA repair activity

Protective properties of human interferons against physical and chemical mutagens have been described earlier. This work was aimed at detecting an optimum of protective action of interferons in human fibroblasts using two criteria: the number of single-strand DNA breaks formed and the index of DNA repair synthesis. The protective ability of interferon was shown to be expressed starting after 4 h of cells' pretreatment and proceeding through 40 h in experiments with N-methyl-N-nitro-N-nitrosoquanidin. The phenomenon of stimulation of DNA repair synthesis in human cells pretreated with interferon proceeded even after replating cells during 8 h in the experiments with UV irradiation.     

Actinomycin D in low concentrations binds uniformly to human chromosomes

The binding of actinomycin D (actD) to fixed human metaphase chromosomes was studied by using autoradiography with [3H]actD and indirect immunofluorescence with a specific anti-actD antibody. At concentrations of 0.01 and 0.1 micrograms/ml there was a uniform distribution of drug along the chromosomes as observed by both methods. This is the first study to date characterizing actD binding at such low concentrations to human chromosomes. Since actD intercalates into the DNA helix with GC specificity, our observations indicate that detectable differences in base composition along the lengths of human chromosomes are minimal.     

Induction by nitrogen and low temperature of storage-protein synthesis in poplar trees exposed to long days

The synthesis of storage proteins in trees of poplar (Populus x canadensis Moench) could not only be induced by a shift from long-day to short-day conditions but also by either a low-temperature treatment or by nitrogen feeding under continuous long-day conditions. The synthesis of the protein did not depend on the cessation of growth and the formation of a terminal bud. The accumulation of the storage protein was in all cases preceded by a drastic increase in the level of the corresponding mRNA.Abbreviations cDNA copy DNA - kDA kilodalton