Studies on the genotoxicity of asataf (acephate), an organophosphate insecticide, in a mammalian in vivo system

The genotoxic potential of asataf (acephate) was evaluated by a battery of in vivo tests: bone marrow chromosome aberrations, micronucleus, sperm-shape abnormality and dominant lethal tests in mice. A significant enhancement in the percentage of chromosome aberrations was noticed in 3 doses, 3 routes and 3 h after asataf treatment of groups of mice as well as in chronic (sub-acute) treatment. A significant difference in the occurrence of micronuclei was found only at the highest dose whereas all the results of the sperm-shape abnormality test were highly significant. In the dominant lethal mutagenicity assay only the result (dead implants) of a single week (3rd) with the higher dose differed significantly from control. On the basis of the present in vivo results in mouse test systems asataf may be considered to be a potential mutagen.     

Evaluation of the cytogenetic damage induced by the organophosphorous insecticide acephate

The organophosphorous insecticide acephate was tested for its ability to induce in vitro cytogenetic effect in human peripheral lymphocytes by using the chromosomal aberrations (CAs), sister chromatid exchange (SCE) and micronuclei (MN) assay. The level of nuclear DNA damage of acephate was evaluated by using the comet assay. Concentrations of 12.5, 25, 50, 100 and 200 μg mL⁻¹ of acephate were used. All concentrations of acephate induced significant increase in the frequency of CAs and in the formation of MN dose dependently (r = 0.92 at 24 h, r = 0.95 at 48 h for CAs, r = 0.87 for MN). A significant increase was observed in induction of SCE at 50, 100 and 200 μg mL⁻¹ concentrations during 24 h treatment and at all concentrations (except 12.5 μg mL⁻¹) during 48 h treatment period in a dose-dependent manner (r = 0.84 at 24 h, r = 0.88 at 48 h). Acephate did not affect the replicative index and cytokinesis-block proliferation index (CBPI). However, it significantly decreased the mitotic index at all three highest concentrations (50, 100, 200 μg mL⁻¹) for 24 h treatment and at all concentrations (except 12.5 μg mL⁻¹) for 48 h treatment, dose-dependently (r = 0.94 at 24 h, r = 0.92 at 48 h). A significant increase in mean comet tail length was observed at 100 and 200 μg mL⁻¹ concentrations compared with negative control in a concentration-dependent manner (r = 0.94). The mean comet tail intensity was significantly increased at only 200 μg mL⁻¹ concentration. The present results indicate that acephate is a clastogenic, cytotoxic agent and it causes DNA damage at high concentrations in human lymphocytes in culture.     

Detection of organophosphate and carbamate pesticides in vegetable samples by a photothermal biosensor

Previously developed photothermal biosensor was optimised by determining the most suitable enzyme substrate (acetylthiocholine iodide) and the optimal carrier buffer (0.05 M phosphate buffer, pH 8.0). Excitation laser operating at 488 nm and 120 mW power provided the highest biosensor sensitivity. The biosensor was tested for detection of toxic organophosphate and carbamate compounds present in samples of salad, iceberg lettuce, and onion. Sufficient sensitivities to different pesticides (carbofuran, propamocarb, oxydemeton-methyl and parathion-ethyl) were achieved without time-consuming sample preparation procedures. The results show good agreement with the concentrations of pesticides determined with standard GC-MS detection method. The developed photothermal biosensor offers new low cost means to detect low concentrations of pesticides in vegetable samples with high throughput and little or no sample pretreatment.     

Effect of diazinon, an organophosphate insecticide, on plasma lipid constituents in experimental animals

There has been increasing interest in studying the various effects of organophosphate insecticides in humans and experimental animals. Only a few data are available on the effect of the organophosphate insecticide, diazinon, on lipid metabolism. The aim of this study was to evaluate the effect of diazinon on plasma lipid constituents in mammalian animals. The plasma levels of total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), and phospholipids (PL) were measured in albino rats that were orally treated with a single dose of diazinon at a level of LD(50) or with repeated daily doses at the levels of 1/2, 1/8, and 1/32 LD(50) for 2, 8, and 32 days, respectively. After a 24 h post-treatment with a single LD(50) dose of diazinon, TC was not significantly changed, the HDL-C and PL levels were significantly decreased, but the LDL-C and TG levels were significantly increased. Separate daily oral administrations of diazinon at 1/2 LD(50), 1/8 LD(50), and 1/32 LD(50) doses resulted in a significant decrease in HDL-C and PL, with no significant change in TG. The LDL-C levels were significantly increased and TC showed no significant change with 1/2 LD(50) and 1/32 LD(50) doses of diazinon, whereas a significant decrease in the levels of TC, HDL-C, as well as LDL-C, was observed with the 1/8 LD(50) dose. These data suggest that diazinon may interfere with lipid metabolism in mammals.     

Metabolism of beta-methylaspartate by a pseudomonad

A bacterium was isolated from soil which utilizes threo-beta-methyl-l-aspartate, certain other amino acids, and a variety of organic substances as single energy sources. It is, or closely resembles, Pseudomonas putida biotype B. The ability of this organism to rapidly decompose such amino acids is dependent on inducible enzyme systems. Dialyzed cell-free extracts of this bacterium metabolize beta-methylaspartate only when catalytic amounts of alpha-ketoglutarate, or pyruvate, and pyridoxal phosphate are also present. The main products formed from beta-methylaspartate under these conditions are alpha-aminobutyrate, carbon dioxide, and alpha-ketobutyrate. When l-aspartate is substituted for beta-methylaspartate in this system, it is converted mainly to alanine and carbon dioxide. beta-Methyloxalacetate is decarboxylated, and the resulting alpha-ketobutyrate is converted enzymatically in the presence of glutamate to alpha-aminobutyrate which accumulates. The added keto acids are converted, in part, to the corresponding amino acids probably by transamination. The data indicate that beta-methylaspartate is converted to alpha-aminobutyrate, and aspartate to alanine, by a circuitous transamination-beta-decarboxylation-transamination sequence rather than by a direct beta-decarboxylation.     

Degradation of 2-fluorobenzoate by a pseudomonad

Pseudomonas (spp), isolated from a complex petrochemical sludge, was able to utilize 2-fluorobenzoate as its sole source of carbon and energy. At the end of the growth phase, about 42% of the organically bound fluoride was released. Catechol, 3-fluorocatechol, and 6-fluorodihydrodihydroxybenzoate were confirmed as intermediates by chromatographic and spectral analyses. During 2-fluorobenzoate metabolism, fluoride is eliminated before the aromaticity of the ring is lost. Twofold higher levels of catechol 1,2-oxygenase were detected in 2-fluorobenzoate-grown cells compared with cells grown on benzoate. When used as assay substrates, 3-chlorocatechol showed less catechol 1,2-oxygenase activity than catechol or 4-chlorocatechol. The ability to degrade 4-fluorobenzoate could be transferred toPseudomonas (spp) by the conjugal transfer of plasmid pWR1 fromPseudomonas sp. B13.     

Degradation of triphenyltin by a fluorescent pseudomonad

Triphenyltin (TPT)-degrading bacteria were screened by a simple technique using a post-column high-performance liquid chromatography using 3,3',4',7-tetrahydroxyflavone as a post-column reagent for determination of TPT and its metabolite, diphenyltin (DPT). An isolated strain, strain CNR15, was identified as Pseudomonas chlororaphis on the basis of its morphological and biochemical features. The incubation of strain CNR15 in a medium containing glycerol, succinate, and 130 microM TPT resulted in the rapid degradation of TPT and the accumulation of approximately 40 microM DPT as the only metabolite after 48 h. The culture supernatants of strain CNR15, grown with or without TPT, exhibited a TPT degradation activity, whereas the resting cells were not capable of degrading TPT. TPT was stoichiometrically degraded to DPT by the solid-phase extract of the culture supernatant, and benzene was detected as another degradation product. We found that the TPT degradation was catalyzed by low-molecular-mass substances (approximately 1,000 Da) in the extract, termed the TPT-degrading factor. The other fluorescent pseudomonads, P. chlororaphis ATCC 9446, Pseudomonas fluorescens ATCC 13525, and Pseudomonas aeruginosa ATCC 15692, also showed TPT degradation activity similar to strain CNR15 in the solid-phase extracts of their culture supernatants. These results suggest that the extracellular low-molecular-mass substance that is universally produced by the fluorescent pseudomonad could function as a potent catalyst to cometabolite TPT in the environment.     

The metabolism of nitrilotriacetate by a pseudomonad

1. An organism that grows on nitrilotriacetate as sole source of carbon and energy was isolated in pure culture and was identified as a pseudomonad. 2. Cell-free extracts of the nitrilotriacetate-grown pseudomonad contain an enzyme that catalyses the NADH-and O(2)-dependent oxidation of nitrilotriacetate to iminodiacetate and glyoxalate. This enzyme is absent from extracts of glucose-grown cells. 3. Compared with growth on glucose, growth on nitrilotriacetate results in increased activities of enzymes of glycine and serine metabolism, namely serine hydroxymethyltransferase, glycine decarboxylase, serine-oxaloacetate aminotransferase and hydroxypyruvate reductase. 4. Cell-free extracts of the nitrilotriacetate-grown organism contain the enzyme glyoxalate carboligase and, when supplemented with NADH, Mg(2+) and thiamin pyrophosphate, can catalyse the anaerobic conversion of glyoxalate into glycerate. 5. These results are incorporated in a scheme which shows the oxidative metabolism of nitrilotriacetate by the successive removal of C(2) units to form 2mol of glyoxalate and 1mol of glycine per mol of nitrilotriacetate degraded. The glyoxalate and glycine are then both metabolized to glycerate by separate pathways, via tartronic semialdehyde and serine respectively. The role of this scheme in the growth of the organism on nitrilotriacetate is discussed.     

Microbial cometabolism of sucralose,a chlorinated disaccharide,in environmental samples

During the rapid mineralization in soil of sucralose (4-chloro-4-deoxy-,D-galactopyranosyl-1,6-dichloro-1,6-dideoxy-,D-fructofuranoside), a metabolic product was formed that appears to be the corresponding unsaturated aldehyde. During the slow and incomplete mineralization of sucralose in lake water, which was not increased by the addition of nitrogen and phosphorus, the same compound was produced. That product was further metabolized by microorganisms in lake water and soil. Mineralization was also slow in sewage under aerobic conditions, but organic products were not detected. Little or no CO₂ was formed from the disaccharide in flooded soil or anaerobic sewage. Bacteria in culture did not use sucralose as a carbon source but did convert it to the presumed unsaturated aldehyde, 1,6-dichloro-1,6-dideoxy-D-fructose and possibly the uronic acid of sucralose. Sucralose carbon was not incorporated into cells of two sucralose-metabolizing bacteria or the microbial biomass of sewage or lake water. The chlorinated disaccharide was slowly metabolized by a galactose oxidase preparation. It is concluded that the chlorinated sugar is acted on microbiologically by cometabolism.     

Utilization of cutin by a pseudomonad isolated from soil

Summary A Pseudomonas sp., which has been isolated from orchard soil, is able to utilize cutin as a sole source of carbon. Products obtained from the culture filtrate corresponded to that obtained by alkaline hydrolysis of cutin.     

Cell differentiation of Proteus mirabilis is initiated by glutamine, a specific chemoattractant for swarming cells

Swarming by Proteus mirabilis involves differentiation of typical short vegetative rods into filamentous hyper-flagellated swarm cells which undergo cycles of rapid and co-ordinated population migration across surfaces and exhibit high levels of virulence gene expression. By supplementing a minimal growth medium (MGM) unable to support swarming migration we identified a single amino acid, glutamine, as sufficient to signal initiation of cell differentiation and migration. Bacteria isolated from the migrating edge of colonies grown for 8h with glutamine as the only amino acid were filamentous and synthesized the characteristic high levels of flagellin and haemolysin. In contrast, addition of the other 19 common amino acids (excluding glutamine) individually or in combination did not initiate differentiation even after 24 h, cells remaining typical vegetative rods with basal levels of haemolysin and flagellin. The glutamine analogue γ-glutamyl hydroxamate (GH) inhibited swarming but not growth of P. mirabilis on glutamine MGM and transposon mutants defective in glutamine uptake retained their response to glutamine signalling and its inhibition by GH, suggesting that differentiation signalling by glutamine may be transduced independently of the cellular glutamine transport system. Levels of mRNA transcribed from the haemolysin (hpmA) and flagellin (fliC) genes were low in vegetative cells grown on MGM without glutamine or with glutamine and GH, but were specifically increased c. 40-fold during glutamine-dependent differentiation. In liquid glutamine—MGM cultures, differentiation to filamentous hyper-flagellated hyper-haemorytic swarm cells occurred early in the exponential phase of growth, and increased concomitantly with the concentration of glutamine from a 0.1 mM threshold up to 10 mM. Differentiation in liquid culture was completely inhibited by GH but was further stimulated c. 30% in the absence of GH by the viscosity agent polyvinylpy-rollidone (PVP). Chemotaxis assays of bacterial cells in which the viscosity of liquid media was varied by PVP to allow either swimming or swarming motility demonstrated that glutamine was chemoattractive specifically to differentiated swarming cells.     

Metabolism of 3-chloro-, 4-chloro-, and 3,5-dichlorobenzoate by a pseudomonad.

Pseudomonas sp. WR912 was isolated by continuous enrichment in three steps with 3-chloro-, 4-chloro-, and finally 3,5-dichlorobenzoate as sole source of carbon and energy. The doubling times of the pure culture with these growth substrates were 2.6, 3.3, and 5.2 h, respectively. Stoichiometric amounts of chloride were eliminated during growth. Oxygen uptake rates with chlorinated benzoates revealed low stereospecificity of the initial benzoate 1,2-dioxygenation. Dihydrodi-hydroxybenzoate dehydrogenase, catechol 1,2-dixoygenase, and muconate cycloisomerase activities were found in cell-free extracts. The ortho cleavage activity for catechols appeared to involve induction of isoenzymes with different stereospecificity towards chlorocatechols. A catabolic pathway for chlorocatechols was proposed on the basis of similarity to chlorophenoxyacetate catabolism, and cometabolism of 3,5-dimethylbenzoate by chlorobenzoate-induced cells yielded 2,5-dihydro-2,4-dimethyl-5-oxo-furan-2-acetic acid.     

Metabolism of 3-chloro-, 4-chloro-, and 3,5-dichlorobenzoate by a pseudomonad.

Pseudomonas sp. WR912 was isolated by continuous enrichment in three steps with 3-chloro-, 4-chloro-, and finally 3,5-dichlorobenzoate as sole source of carbon and energy. The doubling times of the pure culture with these growth substrates were 2.6, 3.3, and 5.2 h, respectively. Stoichiometric amounts of chloride were eliminated during growth. Oxygen uptake rates with chlorinated benzoates revealed low stereospecificity of the initial benzoate 1,2-dioxygenation. Dihydrodi-hydroxybenzoate dehydrogenase, catechol 1,2-dixoygenase, and muconate cycloisomerase activities were found in cell-free extracts. The ortho cleavage activity for catechols appeared to involve induction of isoenzymes with different stereospecificity towards chlorocatechols. A catabolic pathway for chlorocatechols was proposed on the basis of similarity to chlorophenoxyacetate catabolism, and cometabolism of 3,5-dimethylbenzoate by chlorobenzoate-induced cells yielded 2,5-dihydro-2,4-dimethyl-5-oxo-furan-2-acetic acid.     

Detection of Naegleria fowleri cysts in environmental samples by using a DNA probe

Abstract In this study we tried to detect DNA Naegleria fowleri in artificially contaminated environmental samples, with or without sediments, containing 10⁴ cysts of this pathogenic amoeba. We used two assays to extract DNA from samples: first, direct DNA extraction, which gave positive results only for water samples without sediment; second, DNA extraction after sample incubation on agar plates, which allowed us to remove amoeba growing out of the sediments, and which gave positive results for all samples, even those initially with sediments (5, 500 or 500 mg). Thus, this molecular identification appears as a powerful tool to investigate N. fowleri growth in environmental samples.     

Oxidative damage to human red blood cells treated with chlorfenvinphos, an organophosphate insecticide (in vitro)

Chlorfenvinphos (CFVF) is an organophosphorus insecticide, which was used to control insect pest on livestock and household pests such as flies, fleas, and mites. The molecular basis of toxic properties of CFVF in animals has been insufficiently studied. Blood can transport oxygen and nutrients as well as toxic compounds. Xenobiotics can enter to red blood cells and cause damage. Therefore, investigation of the toxicity of different compounds to erythrocytes is very important. The purpose of the present experiment was to evaluate the effect of this compound on human erythrocytes. We have evaluated the hemolysis, hemoglobin oxidation (met-Hb formation) and lipid peroxidation in human erythrocytes. Moreover, the changes in the level of reactive oxygen species (ROS) were assessed using flow cytometry as well as those in morphological changes of erythrocytes using phase contrast microscopy. This study describes the interaction of low concentrations of CFVF with human erythrocytes as well as the concentrations, which may enter human organism as a result of acute poisoning (0.5–250 μM). It was shown that CFVF only at high concentration induced changes in human erythrocytes. We have observed hemolysis (at 250 μM), changes in morphological parameters including echinocytes formation (at 250 μM), as well as increase in lipid peroxidation in erythrocytes (at 250 μM), ROS formation (at 100 μM) in red blood cells treated 1 hour with CFVF. Additionally, CFVF after 4 h of incubation oxidized hemoglobin, however, to a lower degree.     

Production of the siderophore aerobactin by a halophilic pseudomonad.

A bacterial strain, isolated from a cyanobacterial culture, was identified as Pseudomonas sp. strain X40. Under iron-limiting conditions, the Pseudomonas sp. produced aerobactin, a dihydroxamate siderophore previously found only in the family Enterobacteriaceae. Aerobactin was identified by electrophoretic mobility, spectrophotometric titration, proton nuclear magnetic resonance spectroscopy, mass spectrometry, acid hydrolysis, and biological activity. Aerobactin was used as a siderophore in the Pseudomonas sp. and Escherichia coli. Two iron-repressed outer membrane proteins were observed in the Pseudomonas sp., neither of which had electrophoretic mobility identical to that of the aerobactin outer membrane receptor protein from E. coli. DNA hybridization assays showed no hybridization to the aerobactin genes from the E. coli plasmid pColV, indicating that the genetic determinants for aerobactin production by Pseudomonas strain X40 differ substantially from those found in the archetypic enteric plasmid pColV-K30.