The study of the Ca2+ role in cytotoxic response of human cells in culture to the action of xenobiotics

The role of Ca2+ in mechanisms of cell death, necrosis and apoptosis is diverse and generally recognized. The purpose of this work was to study Ca2+ participation in a cytotoxic response of human cultured cells in the presence of toxic concentrations of cationic antiseptic substance poly(hexamethylene guanidine), anionic surfactant SDS and monomeric methyl methacrylate (a component of bone cement applied in surgery). Human cell line U-937 grown in suspension was used for this study. A fluorescent probe chlortetracycline was used, as an indicator of Ca2+ transport through biologic membranes. Our results show that weakly toxic concentrations of xenobiotics under study, close to the minimum toxic doses, nearly always provoke a fair but statistically significant drop in Ca2+ binding by cells. At the same time, higher toxic doses lead to significant increase in Ca2+ influx. The latter event well compares with the majority of literary data, while the mentioned decrease in Ca2+ influx at low toxic concentrations of xenobiotics presumably correlates with the initial stage of acute cytotoxic response, accompanied by a metabolic activation and enhanced resistance of cells to injuring stimuli, demonstrated by the authors elsewhere. In parallel, a possible effect of Ca(2+)-channel antagonist nifedipine was explored under conditions of cytotoxic response of cell lines U-937, A-549 and human embryonic lung fibroblasts to poly(hexamethylene guanidine). Nifedipine (10 microM) was introduced in the incubation medium simultaneously with the toxic agent, and the cells were further maintained for 5 or 24 h in culture; their viability was monitored with the microtetrasolium test or by assessment of LDH leakage into the incubation medium. The effect of nifedipine proved to be dual, depending on the applied concentration of toxic agent: at low toxic concentrations the improvement of viability could be noticed, while at more pronounced toxic doses aggravation of viability was evident. From our point of view the explanation of this result could be the following. In weakly toxic conditions, as in intact cells, Ca2+ influx is brought about by specific mechanisms, mainly through Ca(2+)-channels, that is why nifedipine partly abolishes Ca(2+)-dependent cytotoxic response. At high concentrations, cell plasma membrane is directly damaged by toxic agent, Ca2+ enters cells mainly non-specifically, so that Ca2+ antagonist cannot protect cell injury. The reason of toxic effect aggravation by nifedipine in these conditions is still waiting for its explanation.     

Toxicity of quinoline alkaloids to cultured Cinchona ledgeriana cells

The toxicity of Cinchona alkaloids to cell cultures of C. ledgeriana has been studied in relation to alkaloid uptake and possibilities for selecting high-yielding cell lines. The most toxic, quinine, was completely toxic at 5.5 mM. Both quinine and quinidine were more toxic than their unmethoxylated precursors, cinchonidine and cinchonine. The permanently-charged metho-chlorides of quinine and cinchonidine were less toxic than the parent alkaloids, despite showing similar accumulation ratios in 5-day uptake experiments at sub-toxic concentrations (ca 1.7mM). The toxicity of the natural quinoline alkaloids appears to be a non-specific effect which may be caused by intracellular alkalinisation following uptake of the uncharged bases. The use of precursors of quinine and quinidine as toxic agents for the selection of cell lines with enhanced quinine and quinidine production is ruled out by the lower toxicity of these precursors and by the correlation of an apparently non-specific toxicity with uptake.     

Topographic analysis of the toxic Gef protein from Escherichia coli

The chromosomal gef gene of Escherichia coli is a member of the gef gene family which encodes strongly toxic proteins of about 50 amino acids. We demonstrate here that the Gef protein is detectable by anti-peptide antibodies. Furthermore, we show that Gef is anchored in the cytoplasmic membrane by the N-terminal part of the protein, and that the C-terminal part is localized in the periplasm in a dimeric form with at least one disulphide bond. By mutagenesis of gef it is shown that the periplasmic portion of Gef encodes the toxic domain and that the dimerization of Gef is not essential for the toxic effect.     

球形芽孢杆菌TS—1灭蚊毒蛋白的酶联免疫吸附测定

Bacillus sphaericus Ts-1 Mosquito larvicidal toxins 42 k Da and 43 k Da were isolated by Sephadex G-200 chromatography. Three strains of highly toxic B. sphaericus and two non toxic strains were screened for toxic proteins using ELISA. The lowest detectable toxin level was 1.56 X 10(-5) mg/ml. Non toxic strains did not produce antigens reacting to either the 42 kDa or the 43 kDa antibodies. Ts-1 cultures were examined at 12 and 24 h by LC50 bioassay against Culex pipiens. The LC50's at 12 h and 24 h were 0.71 ppm and 0.154 ppm, respectively, i.e., the toxin level at 24 h was 4.6 times the level at 12 h. ELISA tests established total toxin at 0.049 mg/ml and 0.225 mg/ml at 12 h and 24 h, respectively, confirming the LC50 study.     

Metabolic adaptation of Pseudomonas pseudoalcaligenes CECT5344 to cyanide: role of malate-quinone oxidoreductases, aconitase and fumarase isoenzymes

In general, the biodegradation of a toxic compound by a micro-organism requires the concurrence of, at least, two features in the biological system: first, the capability of the micro-organism to metabolize the toxic compound, and secondly, the capacity to resist its toxic effect. Pseudomonas pseudoalcaligenes CECT5344 is a bacterium used in the biodegradation of cyanide because it is capable to use it as a nitrogen source. The present review is mainly focused on the putative role of iron-containing enzymes of the tricarboxylic acid cycle in cyanide resistance by P. pseudoalcaligenes CECT5344.     

Remediation of Clay Soils Contaminated with Potentially Toxic Elements: The Santo Amaro Lead Smelter,Brazil, Case

The remediation of clayey soil contaminated by potentially toxic elements is a challenging task, especially because of the low permeability and strong affinity of such toxic elements, which prevent the effective use of low-cost in situ remediation methods. The clay soil from the region of the former Santo Amaro primary lead smelter has high concentrations of potentially toxic elements, especially Pb, Cd, Sb, and Zn. This study presents a preliminary evaluation of remediation by soil washing and thermal stabilization, which can support clean-up initiatives for this site. The treatment results indicate that soil washing using EDTA can be an effective method to clean up the soil; however, the solid-liquid separation step by filtration is slow. Soil thermal treatment at temperatures higher than 800°C in an oxidant atmosphere results in the formation of ceramic structure because of the high smectite content, which stabilizes the potentially toxic elements. The estimated cost for remediation by soil excavation, replacement, and stabilization, of the critically contaminated site at the Santo Amaro region is estimated at about 20 to 30 million US$.     

Toxicity of secondary compounds to the seed-eating larvae of the bruchid beetle Callosobruchus maculatus

By incorporating various secondary compounds in the normal diet of larval Callosobruchus maculatus bruchids, we show that the effects of any particular compound are dosage-dependent. Alkaloids are generally the most toxic of the compounds tested. Non-protein amino acids are more toxic than protein amino acids but the latter can be toxic at 1 and 5% incorporation in the diet. The non-protein amino acid homoarginine has a salutary effect on larval survival at low concentrations. A variety of other secondary compounds found in seeds are toxic at various levels representative of those levels found in seeds in nature, and for all secondary compounds tested a 0.1–5% incorporation in the diet often has a detrimental effect on production of adult beetles. We conclude that many of the secondary compounds found in seeds are likely to be toxic to at least some animal, and thus are likely to be responsible at least in part for the extreme host-specifity shown by seed-eating insects.     

Microbial degradation of pollutants at high salt concentrations

Though our knowledge on microbial degradation of organic pollutants at high salt concentrations is still limited, the list of toxic compounds shown to be degraded or transformed in media of high salinity is growing. Compounds transformed aerobically include saturated and aromatic hydrocarbons (by certain archaeobacteria), certain aromatic compounds, organophosphorus compounds, and formaldehyde (by halotolerant eubacteria). Anaerobic microbial transformations of toxic compounds occurring at high salt concentrations include reduction of nitroaromatic compounds, and possibly transformation of chlorinated aromatic compounds.     

Inhibitory effect of a lethal toxic fragment of staphylococcal alpha-toxin on cyclic AMP-dependent protein kinase activity.

The effect of a lethal toxic fragment of staphylococcal alpha-toxin on the activity of adenosine 3',5'-monophosphate(cyclic AMP)-dependent protein kinase was examined. 1. The lethal toxic fragment produced a dose-dependent decrease in both the binding of cyclic AMP to the regulatory subunit and phosphorylation activity of cyclic AMP-dependent protein kinase obtained from rabbit skeletal muscles up to a plateau at a 50% inhibitory effect. The decrease in the activity of protein kinase observed with low doses of the lethal toxic fragment (0.1 microM) resulted from a competitive inhibition, probably by its interaction with the cyclic AMP-binding site in the regulatory subunit molecule. 2. The effects of a lethal toxic fragment and epinephrine on the cyclic AMP level and protein kinase activity were investigated in the perfused rabbit heart slices. The lethal toxic fragment attenuated the stimulation of cyclic AMP-dependent protein kinase activity ratio by epinephrine. 3. It is suggested that the specific action of a lethal toxic fragment on the cellular membrane enzymes may be attributable to the inhibition of the cyclic AMP-dependent protein kinase activity.     

生活污水对稀有(鱼句)鲫的毒性效应研究

利用稀有鲫个体小、性成熟时间短、在实验室控制条件下容易饲养等特点 ,将其作为一种毒性实验材料 ,从形态学和组织学水平对生活污水可能产生的毒性效应进行了初步研究。研究结果表明 ,在早期生命阶段 ,高浓度的生活污水对稀有鲫有急性毒性效应。受排污口污水暴露的稀有鲫鱼卵根本无法孵化 ;对处于性腺发育期稀有鲫的暴露则引起肝脏细胞水平上的毒性效应 ,且随污水浓度的增高 ,对鱼体性发育的阻碍效应也越强 ,而整个生命期即使仅暴露于低浓度的生活污水中 ,其个体发育和性发育也均受到影响 ,肝细胞也表现出强烈的毒性效应     

Identifying polyglutamine protein species in situ that best predict neurodegeneration

Polyglutamine (polyQ) stretches exceeding a threshold length confer a toxic function to proteins that contain them and cause at least nine neurological disorders. The basis for this toxicity threshold is unclear. Although polyQ expansions render proteins prone to aggregate into inclusion bodies, this may be a neuronal coping response to more toxic forms of polyQ. The exact structure of these more toxic forms is unknown. Here we show that the monoclonal antibody 3B5H10 recognizes a species of polyQ protein in situ that strongly predicts neuronal death. The epitope selectively appears among some of the many low-molecular-weight conformational states assumed by expanded polyQ and disappears in higher-molecular-weight aggregated forms, such as inclusion bodies. These results suggest that protein monomers and possibly small oligomers containing expanded polyQ stretches can adopt a conformation that is recognized by 3B5H10 and is toxic or closely related to a toxic species.     

Characterization of extracellular substance of Vibrio anguillarum toxic for rainbow trout and mice

An extracellular toxic substance was separated from the cell-free culture filtrate of Vibrio anguillarum (strain NCMB571). Two fractions (GI and GII + III) obtained by Sephadex G-200 chromatography following DEAE-cellulose chromatography were lethal to rainbow trout and mice. Material separated from the GI fraction by Sepharose 4B affinity chromatography (GI-A fraction) was lethal to these animals. By sodium dodecylsulfate polyacrylamide gel electrophoresis, the GI and GI-A fractions were found to be composed of components with molecular weights of 44K and 34K, and 44K, respectively. The 44K protein band was associated with carbohydrate. Peripheral vascular disorder was observed in fish and mice that died after inoculation with GI or GI-A fraction. The toxic substance was sensitive to potassium periodate but was resistant to trypsin and acetone. Heat inactivation of the toxic substance was almost complete at 100 C for 20 min and complete at 121 C for 20 min. The toxic activity was not associated with hemolytic or proteolytic activity. Homologous antitoxin completely neutralized the toxic activity.     

Effects of Temperature and Tension on Oxygen Toxicity for the Protozoa of Cryptocercus

SUMMARY. At any given temperature level, the rate of oxygen poisoning increases proportionally with an increase in oxygen tension. But the toxicity of oxygen does not bear a proportional relationship to temperature. At a constant low tension, it is more toxic at low temperatures than at high ones and, at a constant high tension, it is less toxic at low temperatures than at high ones.     

Effect of the replacement of oxygen with sulphur in the cresol molecule on toxicity to goldfish

Summary A study was made of the toxicity of the three tolyl mercaptans with respect to concentration and survival time at 27 °C, and the results were compared with each other, as well as with phenol, rotenone, and the corresponding cresols. Goldfishes of the same lot, each weighing 6.0 to 7.5 g., were used as the test animals.The type of toxic action of the tolyl mercaptans was found to be similar to that of phenyl mercaptan, but different from that of the corresponding cresols.According to the minimum product of concentration and survival time, which measures toxicity at its range of most powerful action,m-tolyl mercaptan is the least toxic of the isomers. It is about four times as toxic, the ortho compound is nearly five times as toxic and the para compound is about eight and one-half times as toxic as phenol. Rotenone is about 85, 70, and 40 times as toxic as the respective mercaptans.Compared according to the same criterion, the tolyl mercaptans bear the following ratios of toxicity: meta, 1.00; ortho, 1.19; and para, 2.19. These are practically the same relationships as were found for the corresponding cresols.Essentially the same relationships are shown by other methods of comparison of the same data, which express relative toxicity under conditions in the neighborhood of its maximum power with respect to concentration and time. Methods emphasizing the effect of the threshold of toxicity, however, lead to greater expansion of the toxic ratios.The replacement of the oxygen atom of the cresol molecule with sulphur causes a fourfold increase in toxicity. A similar chemical relationship in the phenyl homologues, however, has been found in a previous study to correspond to a sevenfold difference in toxicity to goldfish of much smaller size.     

Effect of Toxic Compounds of Exserohilum turcicum on Chlorophyll Content, Callus Growth and Cell Viability of Susceptible and Resistant Inbred Lines of Maize

Toxicity of toxic compounds in the culture filtrates of Exserohilum turcicum towards seed germination, inhibition of shoot and root growth, callus growth and on reduction in chlorophyll content and cell viability of maize was studied. There was no germination in unautoclaved extracts at 100% (undiluted) and 50% concentrations, whereas germination was normal at all the concentrations of autoclaved toxic compounds. Inhibition of root growth was higher compared with that of shoot growth in all the test preparations of toxic compounds. Reduction in total chlorophyll and also chlorophyll a and b contents of disease resistant (KWTC-17) and susceptible (DK-4) inbreds occurred at 50, 25 and 10% concentrations of toxic compounds, with maximum reduction in the susceptible compared with the resistant inbred. Chlorophyll a was found to be more sensitive, showing 7.2% reduction in content in susceptible inbred even at 5% toxin concentration. Callus cultures/cell suspensions derived from coleotible nodes of DK-4 were more sensitive compared with that of KWTC-17 in all the test concentrations of toxic compounds. Callus growth of susceptible inbred DK-4 was inhibited by 32.7% compared with 15.31% of resistant inbred KWTC-17 at 5% concentration of toxic compounds. Cell viability of resistant and susceptible inbreds was 62.0% and 20.2% at 10% toxic concentration, respectively. Spore germination assay with four fungal species or leaf puncture assay were not found useful.     

Research needs to improve risk assessment of fiber toxicity

Risk characterization of exposure to toxic compounds requires information on the intrinsic toxic properties, including toxic mechanism and toxicokinetics, on dose response at the most critical targets for identification of the NOEL or for extrapolation from high to low dose, and on human exposure. Abundant information is available on the intrinsic properties of MMMF, on the three D's (dose, dimension, durability) and on the toxic mechanisms. However, only a few of these studies provide information on the dose response of the effects or of the mechanisms investigated. Moreover, in many cases single high doses exceeding the MTD have been applied and are difficult to interpret for lower exposure scenarios. Risk characterization is further hampered by the still open question whether MMMF are directly genotoxic or induce secondary genotoxicity via inflammation. Finally, there is disagreement about the relevance of animal studies on MMMF for humans and thus about the most rational extrapolation of the dose response of toxic effects observed in animals to man. These deficits are briefly described and discussed from a toxicological point of view.     

Differential Sensitivity of Wheat Embryos against Extracts Containing Toxins of Septoria nodorum: First Steps towards in vitro Selection

As a first step towards the development of an in vitro -selection system for septoria nodorum blotch resistance, wheat embryo culture on media containing extracts from Septoria nodorum was established. Extracts prepared from inoculated wheat grains had a toxic activity. Control extracts from uninoculated grains showed at least a 10-fold lower toxic activity. Two wheat breeding lines susceptible to Septoria nodorum showed reduced growth in the presence of the fungal extract whencompared to a breeding line known to have good resistance in the field. A test with seven additional wheat lines showed a good agreement between field resistance of the ear and embryo resistance. Mellein is one of the toxins produced by Septoria nodorum and was used in pure form for in vitro -selection. It showed toxic effects at 50 μg/ml, a concentration which is about 200-fold higher than the mellein concentration in the diluted extract with embryotoxic activity. This indicates the importance of additional toxic compounds in the crude extract. Mellein acted non-selectively on embryos of the different cultivars.     

Toxicity of L-Tryptophan photoproduct on recombinationless (rec) mutants of Salmonella typhimurium

l-Tryptophan after exposure to black light becomes toxic for recombinationless (rec) mutants of Salmonella typhimurium. Fifty-six radiation-sensitive mutants were screened for sensitivity to the tryptophan photoproduct; the rec and exr (X-ray sensitive) mutants are sensitive, whereas the uvr, hcr, and wild-type strains are resistant. A number of catabolic products of tryptophan and compounds related to tryptophan were screened for toxicity to rec strain; these are nontoxic or far less toxic for rec strains than irradiated l-tryptophan. The toxic photoproduct is relatively stable to drying and basic hydrolysis at 90 C, indicating that it is a stable organic compound, eliminating peroxide as the toxic component. It was also observed that the toxic product is a photooxidation product since it is formed only when l-tryptophan is irradiated in the presence of air.     

Highly conserved toxicity of Saccharomyces cerevisiae Rap1p

Budding yeast ( Saccharomyces cerevisiae ) Rap1p has been expressed in fission yeast ( Schizosaccharo-myces pombe ) under the control of the regulatable fructose bisphosphatase ( fbp ) promoter. When the fbp promoter was derepressed, cells containing the complete RAP1 gene failed to show any significant growth, suggesting that Rap1p is toxic. A derivative of Rap1p that has a temperature-sensitive mutation in the DNA-binding domain was not toxic in cells grown at 37°C, a temperature at which DNA binding by rap1p ^ts is severely inhibited. Removal of a short region downstream of the DNA-binding domain, including a region previously shown to be essential for Rap1p toxicity in budding yeast, also abolished the toxic effect. The toxic effect of Rap1p has therefore been conserved between two distantly related yeasts. In budding yeast, overexpression of Rap1p also caused changes to the lengths of the telomeric repeats. No effects on telomeres were detected in fission yeast.     

Toxicity to Spodoptera exigua and Trichoplusia ni of individual P1 protoxins and sporulated cultures of Bacillus thuringiensis subsp. kurstaki HD-1 and NRD-12

The toxicities to neonate Spodoptera exigua and Trichoplusia ni of lyophilized powders obtained from sporulated liquid cultures (referred to as sporulated cultures) and Escherichia coli-expressed P1 [cryIA(a) cryIA(b) cryIA(c)] protoxins from three-gene strains of NRD-12 and HD-1 of Bacillus thuringiensis subsp. kurstaki were determined by using diet incorporation bioassays. Although sporulated cultures from both strains were more toxic to T. ni than S. exigua, there were no differences in toxicity between NRD-12 and HD-1. Toxicities of the three individual P1 protoxins against S. exigua varied by at least fivefold, with the cryIA(b) protein being the most toxic. These same protoxins varied in toxicity against T. ni by at least 16-fold, with the cryIA(c) protein being the most toxic. However, when tested against either S. exigua or T. ni, there were no differences in toxicity between an NRD-12 P1 protoxin and the corresponding HD-1 P1 protoxin. Comparing the toxicities of individual protoxins with that of sporulated cultures demonstrates that no individual protoxin was as toxic to S. exigua as the sporulated cultures. However, this same comparison against T. ni shows that both the cryIA(b) and cryIA(c) proteins are at least as toxic as the sporulated cultures. Results from this study suggest that NRD-12 is not more toxic to S. exigua than HD-1, that different protein types have variable host activity, and that other B. thuringiensis components are not required for T. ni toxicity but that other components such as spores might be required for S. exigua toxicity.