Inhibition of AChE by single and simultaneous exposure to malathion and its degradation products

In vitro inhibition of bovine erythrocytes acetylcholinesterase (AchE) by separate and simultaneous exposure to organophosphorous insecticide malathion and the transformation products, which are generally formed during the storage or natural as well as photochemical degradation pathways of malathion, was investigated. The increasing concentration of malathion, its oxidation product - malaoxon and isomerisation product - isomalathion inhibited AChE activity in a concentration-dependent manner. The half-maximum inhibitory concentrations (IC(50) values): (3.2 +/- 0.1) x 10(-5) mol/l, (4.7 +/- 0.8) x 10(-7) mol/l and (6.0 +/- 0.5) x 10(-7)mol/l were obtained from the inhibition curves induced by malathion, malaoxon and isomalathion, respectively. However, the products formed due to photoinduced degradation, phosphorodithioic O,O,S-trimethyl phosphorodithioic ester (OOS(S)) and O,O-dimethyl thiophosphate did not noticeably affect the enzyme activity at all investigated concentrations, while diethyl maleate inhibited the AChE activity at concentrations >10 mmol/l. By simultaneous exposure of the enzyme to malaoxon and isomalathion in various concentration combinations the additive effect was achieved by low concentration of inhibitors, while the antagonistic effect was obtained at high concentration (>or= 3 x 10(-7) mol/l) of inhibitors. Inhibitory power of irradiated samples of 1 +/- 10(-5) mol/l malathion can be attributed to the formation of malaoxon and isomalathion, organophosphates about 100 times more toxic than their parent compound, while the presence of non-inhibiting degradation product OOS(S) did not affect the inhibitor efficiency of inhibiting malathion by-products, malaoxon and isomalathion.     

Inhibition of AChE by malathion and some structurally similar compounds

Inhibition of bovine erythrocyte acetylcholinesterase (free and immobilized on controlled pore glass) by separate and simultaneous exposure to malathion and malathion transformation products which are generally formed during storage or through natural or photochemical degradation was investigated. Increasing concentrations of malathion, its oxidation product malaoxon, and its isomerisation product isomalathion inhibited free and immobilized AChE in a concentration-dependent manner. KI, the dissociation constant for the initial reversible enzyme inhibitor-complex, and k3, the first order rate constant for the conversion of the reversible complex into the irreversibly inhibited enzyme, were determined from the progressive development of inhibition produced by reaction of native AChE with malathion, malaoxon and isomalathion. KI values of 1.3 x 10(-4) M(-1), 5.6 x 10(-6) M(-1) and 7.2 x 10(-6)M(-1) were obtained for malathion, malaoxon and isomalathion, respectively. The IC50 values for free/immobilized AChE, (3.7 +/- 0.2) x 10(-4) M/(1.6 +/-0.1) x 10(-4), (2.4 +/- 0.3) x 10(-6)/(3.4 +/- 0.1) x 10(-6)M and (3.2 +/- 0.3) x 10(-6) M/(2.7 +/- 0.2) x 10(-6) M, were obtained from the inhibition curves induced by malathion, malaoxon and isomalathion, respectively. However, the products formed due to photoinduced degradation, phosphorodithioic O,O,S-trimethyl ester and O,O-dimethyl thiophosphate, did not noticeably affect enzymatic activity, while diethyl maleate inhibited AChE activity at concentrations > 10mM. Inhibition of acetylcholinesterase increased with the time of exposure to malathion and its inhibiting by-products within the interval from 0 to 5 minutes. Through simultaneous exposure of the enzyme to malaoxon and isomalathion, an additive effect was achieved for lower concentrations of the inhibitors (in the presence of malaoxon/isomalathion at concentrations 2 x 10(-7) M/2 x 10(-7) M, 2 x 10(-7) M/3 x 10(-7)M and 2 x 10(-7) M/4.5 x 109-7) M), while an antagonistic effect was obtained for all higher concentrations of inhibitors. The presence of a non-inhibitory degradation product (phosphorodithioic O,O,S-trimethyl ester) did not affect the inhibition efficiencies of the malathion by-products, malaoxon and isomalathion.     

Mutagenic and alkylating activities of organophosphate impurities of commercial malathion

The purpose of this study was to determine if 4 major organophosphate impurities of malathion were active as alkylators of nitrobenzylpyridine (NBP) or as mutagens in the Salmonella typhimurium bioassay. Malathion, isomalathion, O,O,O-trimethyl phosphorothioate, O,O,S-trimethyl phosphorothioate, and O,S,S-trimethyl phosphorodithioate produced alkylated NBP at varying rates. In order of increasing NBP reactivity, the compounds ranked: O,O,O-trimethyl phosphorothioate = O,O,S-trimethyl phosphorothioate less than O,S,S-trimethyl phosphorodithioate less than isomalathion = malathion. At 37 degrees C, the most reactive compounds produced an NBP alkylation rate equal to approximately 25% of the rate produced by methyl methanesulfonate, a potent Salmonella mutagen. However, none of the organophosphates were mutagenic in S. typhimurium TA97, TA98 and TA100 when tested by the standard plate-incorporation method or by the preincubation modification of the plate-incorporation method. The possible relationships between NBP reactivity and the biological activities of these organophosphates are discussed.     

Inhibition of AChE by malathion and some structurally similar compounds

Inhibition of bovine erythrocyte acetylcholinesterase (free and immobilized on controlled pore glass) by separate and simultaneous exposure to malathion and malathion transformation products which are generally formed during storage or through natural or photochemical degradation was investigated. Increasing concentrations of malathion, its oxidation product malaoxon, and its isomerisation product isomalathion inhibited free and immobilized AChE in a concentration-dependent manner. K_I, the dissociation constant for the initial reversible enzyme inhibitor-complex, and k₃, the first order rate constant for the conversion of the reversible complex into the irreversibly inhibited enzyme, were determined from the progressive development of inhibition produced by reaction of native AChE with malathion, malaoxon and isomalathion. K_I values of 1.3 × 10^{? 4} M^{? 1}, 5.6 × 10^{? 6} M^{? 1} and 7.2 × 10^{? 6} M^{? 1} were obtained for malathion, malaoxon and isomalathion, respectively. The IC₅₀ values for free/immobilized AChE, (3.7 ± 0.2) × 10^{? 4} M/(1.6 ± 0.1) × 10^{? 4}, (2.4 ± 0.3) × 10^{? 6}/(3.4 ± 0.1) × 10^{? 6} M and (3.2 ± 0.3) × 10^{? 6} M/(2.7 ± 0.2) × 10^{? 6} M, were obtained from the inhibition curves induced by malathion, malaoxon and isomalathion, respectively. However, the products formed due to photoinduced degradation, phosphorodithioic O,O,S-trimethyl ester and O,O-dimethyl thiophosphate, did not noticeably affect enzymatic activity, while diethyl maleate inhibited AChE activity at concentrations > 10 mM. Inhibition of acetylcholinesterase increased with the time of exposure to malathion and its inhibiting by-products within the interval from 0 to 5 minutes. Through simultaneous exposure of the enzyme to malaoxon and isomalathion, an additive effect was achieved for lower concentrations of the inhibitors (in the presence of malaoxon/isomalathion at concentrations 2 × 10^{? 7} M/2 × 10^{? 7} M, 2 × 10^{? 7} M/3 × 10^{? 7} M and 2 × 10^{? 7} M/4.5 × 10^{? 7} M), while an antagonistic effect was obtained for all higher concentrations of inhibitors. The presence of a non-inhibitory degradation product (phosphorodithioic O,O,S-trimethyl ester) did not affect the inhibition efficiencies of the malathion by-products, malaoxon and isomalathion.     

Enantiomeric separation of malathion and malaoxon and the chiral residue analysis in food and environmental matrix

Malathion is a widely used chiral phosphorus insecticide, which has a more toxic chiral metabolite malaoxon. In this work, the enantiomers of malathion and malaoxon were separated by high-performance liquid chromatography-mass/mass (HPLC-MS/MS) with chiral columns using acetonitrile/water or methanol/water as mobile phase, and the chromatographic conditions were optimized. Based on the chiral separation, the chiral residue analysis methods for the enantiomers in soil, fruit, and vegetables were set up. Two pairs of the enantiomers were better separated on CHIRALPAK IC chiral column, and baseline simultaneous separations of malathion and malaoxon enantiomers were achieved with acetonitrile/water (40/60, v/v) as mobile phase at a flow rate of 0.5 mL/min. The elution orders were −/+ for both malathion and malaoxon measured by an optical rotation detector. The chiral residue analysis in soil, fruit, and vegetables was validated by linearity, recovery, precision, limit of detection (LOD), and limit of quantification (LOQ). The LODs and LOQs for the enantiomers of malathion were 1 μg/kg and 3–5 μg/kg and 0.08 μg/kg and 0.20–0.25 μg/kg for malaoxon enantiomers. Good linear calibration curves for each enantiomer in the matrices were obtained within the concentration range of 0.02–12 mg/L. The mean recoveries of the enantiomers of malathion and malaoxon ranged from 82.26% to 109.04%, with RSDs of 0.71–8.63%.The results confirmed that this method was capable of simultaneously determining the residue of malathion and malaoxon in food and environmental matrix on an enantiomeric level.     

Cytogenetic evaluation of malathion-induced toxicity in Sprague-Dawley rats

Malathion is a well known pesticide and is commonly used in many agricultural and non-agricultural settings. Its toxicity has been attributed primarily to the accumulation of acetylcholine (Ach) at nerve junctions, due to the inhibition of acetylcholinesterase (AChE), and consequently overstimulation of the nicotinic and muscarinic receptors. However, the genotoxicity of malathion has not been adequately studied; published studies suggest a weak interaction with the genetic material. In the present study, we investigated the genotoxic potential of malathion in bone marrow cells and peripheral blood obtained from Sprague-Dawley rats using chromosomal aberrations (CAs), mitotic index (MI), and DNA damage as toxicological endpoints. Four groups of four male rats, each weighing approximately 60 ± 2g, were injected intraperitoneally (i.p.) once a day for five days with doses of 2.5, 5, 10, and 20mg/kg body weight (BW) of malathion dissolved in 1% DMSO. The control group was made up of four animals injected with 1% DMSO. All the animals were sacrificed 24h after the fifth day treatment. Chromosome preparations were obtained from bone marrow cells following standard protocols. DNA damage in peripheral blood leukocytes was determined using alkaline single-cell gel electrophoresis (comet assay). Malathion exposure significantly increased the number of structural chromosomal aberrations (CAs) and the percentages of DNA damage, and decreased the mitotic index (MI) in treated groups when compared with the control group. Our results demonstrate that malathion has a clastogenic/genotoxic potential as measured by the bone marrow CA and comet assay in Sprague-Dawley rats.     

Effects of pesticides on cyanobacterium Plectonema boryanum and cyanophage LPP-1

Cyanobacterium Plectonema boryanum IU 594 and cyanophage LPP-1 were used as indicator organisms in a bioassay of 16 pesticides. Experiments such as spot tests, disk assays, growth curves, and one-step growth experiments were used to examine the effects of pesticides on the host and virus. Also, experiments were done in which host or virus was incubated in pesticide solutions and then assayed for PFU. P. boryanum was inhibited by four herbicides: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 1,1-dimethyl-3-(alpha, alpha,alpha-trifluoro-m-tolyl)urea ( Fluometeron ), 2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine (Atrazine), 2-(ethylamino)-4-(isopropylamino)-6-(methylthio)-s-triazine ( Ametryn ). One insecticide, 2-methyl-2-(methylthio)-propionaldehyde O-( methylcarbamoyl )oxime (Aldicarb), also inhibited the cyanobacterium. Two insecticides inactivated LPP-1, O,O-dimethyl phosphorodithioate of diethyl mercaptosuccinate (malathion) and Isotox . Isotox is a mixture of three pesticides: S-[2-( ethylsulfinyl )ethyl]O,O-dimethyl phosphorothioate ( Metasystox -R), 1-naphthyl methylcarbamate ( Sevin ) and 4,4'-dichloro-alpha- (trichloromethyl) benzhydrom ( Kelthane ). Two pesticide-resistant strains of P. boryanum were isolated against DCMU and Atrazine. These mutants showed resistance to all four herbicides, which indicates a relationship between these phototoxic chemicals. The results indicate that P. boryanum may be a useful indicator species for phototoxic agents in bioassay procedures.     

Effects of pesticides on cyanobacterium Plectonema boryanum and cyanophage LPP-1.

Cyanobacterium Plectonema boryanum IU 594 and cyanophage LPP-1 were used as indicator organisms in a bioassay of 16 pesticides. Experiments such as spot tests, disk assays, growth curves, and one-step growth experiments were used to examine the effects of pesticides on the host and virus. Also, experiments were done in which host or virus was incubated in pesticide solutions and then assayed for PFU. P. boryanum was inhibited by four herbicides: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 1,1-dimethyl-3-(alpha, alpha,alpha-trifluoro-m-tolyl)urea ( Fluometeron ), 2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine (Atrazine), 2-(ethylamino)-4-(isopropylamino)-6-(methylthio)-s-triazine ( Ametryn ). One insecticide, 2-methyl-2-(methylthio)-propionaldehyde O-( methylcarbamoyl )oxime (Aldicarb), also inhibited the cyanobacterium. Two insecticides inactivated LPP-1, O,O-dimethyl phosphorodithioate of diethyl mercaptosuccinate (malathion) and Isotox . Isotox is a mixture of three pesticides: S-[2-( ethylsulfinyl )ethyl]O,O-dimethyl phosphorothioate ( Metasystox -R), 1-naphthyl methylcarbamate ( Sevin ) and 4,4'-dichloro-alpha- (trichloromethyl) benzhydrom ( Kelthane ). Two pesticide-resistant strains of P. boryanum were isolated against DCMU and Atrazine. These mutants showed resistance to all four herbicides, which indicates a relationship between these phototoxic chemicals. The results indicate that P. boryanum may be a useful indicator species for phototoxic agents in bioassay procedures.     

Interaction of O,O,S-trimethyl phosphorothioate and O,S,S-trimethyl phosphorodithioate, the impurities of malathion with supercoiled PM2 DNA

The interaction of O,O,S-trimethyl phosphorothioate and O,S,S-trimethyl phosphorodithioate, the impurities found in malathion, with DNA at pH 8.0, was investigated. Supercoiled PM2 DNA was incubated with these compounds at pH 8.0 at 37 degrees C and then the superhelicity of the modified DNA was determined by gel electrophoresis. Both compounds caused unwinding of supercoiled DNA in dose- and incubation time-dependent manner. O,S,S-trimethyl phosphorodithioate was a more potent agent than O,O,S-trimethyl phosphorothioate. At 37 degrees C following 2.0 hours incubation, 100 mM O,S,S-trimethyl phosphorodithioate produced fully unwound DNA, whereas at 200 mM O,O,S-trimethyl phosphorothioate produced 80% unwound DNA following 12 hours' incubation. At the same condition, 5 mM methyl methanesulfonate, a potent alkylating mutagen, produced fully unwound DNA following 1 hour incubation at 5 mM. These results indicated that there were chemical interactions between these agents and DNA. The possibility of the interaction of OOS-TMP being as a covalent intercalation as well as strand nicking was discussed.     

Genotoxicity of imidacloprid in relation to metabolic activation and composition of the commercial product

Imidacloprid is a neonicotinoid insecticide combining excellent efficiency against parasites with low toxicity for mammals. Commercially, it is co-formulated with dimethyl sulfoxide, methylpyrrolidone, propylene carbonate and mineral oil, which can modify its bioavailability and toxicological profile for humans following occupational exposure. A combined in vitro approach employing the comet assay and the micronucleus test was used to assess the genotoxicity of imidacloprid in relation to formulation, metabolic activation and exposure level. Human peripheral blood lymphocytes from unexposed healthy volunteers were treated with imidacloprid (0.2, 2 and 20 μM) and with equimolar concentrations of a commercial product, with and without addition of S9 fraction. Imidacloprid significantly increased the comet score and the frequency of micronuclei only at the highest concentration tested. DNA damage was slightly more severe with the commercial product, and was increased, though not significantly, by metabolic activation. Formation of reactive oxygen species (ROS) does not seem to be involved as a mechanism of genotoxicity, but this result may be explained by the insufficient sensitivity of the 2',7'-dichlorofluorescein diacetate assay at the test concentrations of imidacloprid. These results suggest that at concentrations<20 μM imidacloprid is not genotoxic to human lymphocytes in vitro. Nonetheless, the presence of co-formulants in the commercial product and occupational exposure, along with poor safety procedures, may present an increased risk for DNA fragmentation and chromosomal aberrations.     

Comparative analysis of cytotoxic, genotoxic and antioxidant effects of 2,2,4,7-tetramethyl-1,2,3,4-tetrahydroquinoline and ethoxyquin on human lymphocytes

2,2,4,7-Tetramethyl-1,2,3,4-tetrahydroquinoline (THQ) is a new synthetic compound with potential antioxidant activity. In this study, cytotoxic, genotoxic and antioxidant activities of THQ were studied on human lymphocytes with the use of the trypan blue exclusion assay, the TUNEL method, the comet assay and the micronucleus test. The activities of THQ were compared with those of a structurally similar compound-ethoxyquin (1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline, EQ), which is used in animal feeds as a preservative. Cytotoxic effects of THQ were observed after 1-h treatment at the concentration of 500 microM and after 24-h treatments at the concentrations of 250-500 microM. Although the micronucleus test did not reveal a genotoxic effect of THQ, in the comet assay the statistically significant increase in DNA damage was observed as compared with the control. On the other hand, the protection of human lymphocytes against DNA damage induced by hydrogen peroxide suggests an antioxidant activity of THQ. The comparative analysis of THQ and EQ activities performed in these studies revealed that THQ was less cytotoxic and less genotoxic than EQ. Slightly lower antioxidant activity of THQ was also shown in the comet assay when it was used at the lower studied doses (1-5 microM), but for the highest one (10 microM) its efficiency was similar to that of EQ. In the micronucleus assay THQ was more effective than EQ in protecting the cultured lymphocytes from clastogenicity of H2O2. We believe that THQ is worthy of further detailed studies on its antioxidant properties to confirm its usefulness as a preservative.     

Cytological effects of an organic phosphate pesticide on human cells in vitro.

This investigation was undertaken to determine the radiomimetic effects in vitro of the insecticide guthion [azinophos-methyl; O, O-dimethyl S-(4-oxobenzotriazino-3-methyl) phosphorodithioate] on two human cell lines, diploid (WI-38) and heteroploid (HEp-2). In WI-38 cells, dosages of 120, 140 and 160 microng/ml of guthion were effective in inducing an average chromosome breakage of 0.042, 0.058 and 0.165 respectively per cell. In control cells, the mean number of breaks per cell was 0.011. Chromosome breakages were significantly higher in treated cells. Since guthion prevents cells from entering into mitosis during treatment, the number of mitiotic cells decreases rapidly following treatment with higher concentrations. Similar dosages in treated HEp-2 cells also induced a high incidence of chromosome aberrations, the most common being chromatid breaks and exchanges. Infrequently noted were mild failure of condensation, despiralization, secondary constrictions, gaps, dicentric chromosomes and pulverization. To date, this study reaffirms previous findings of the potentially hazardous, mutagenic effects of guthion.     

Involvement of chromosomally-encoded genes in malathion utilization by Pseudomonas aeruginosa AA112

Malathion is an organophosphate insecticide that has been widely used for both domestic and commercial agricultural purposes. However, malathion has the potential to produce toxic effects in mammalian systems. In this study, Pseudomonas aeruginosa AA 112 which was isolated from soil using enrichment technique could utilize the malathion as a sole carbon source and a source of energy. Pseudomonas aeruginosa AA112 was able to grow in MSMPY medium containing 42.75 mg/ml malathion. However, the optimum concentration of malathion which supported the maximum bacterial growth was found to be 22. 8 mg/ml. Malathion was used as an initial source of energy and carbon when it was found without additional carbon sources (in MSM medium) while it was utilized as second source of energy and carbon in a nutrient-supplemented medium (in MSMPY medium). Moreover, lead acetate test indicated that malathion was first attacked at a sulphur site 1-2 hours after the start of incubation. TLC and IR analysis indicated that malathion was completely degraded into diethyl succinate, hydrogen sulphide and phosphates. Therefore a malathion degradation pathway was proporsed. The degradation of malathion is attributed to the genes located on the chromosome and at least three proteins of high molecular size might be involved in malathion utilization. Bacteria able to use malathion as a food source or metabolize its residues in the environment to inactive, less toxic, and harmless compounds, could be used in bioremediation of an environmental pollution caused by the pesticide.     

In vivo genotoxicity of the pyrethroid pesticide beta-cyfluthrin using the comet assay in the fish Bryconamericus iheringii

Environmental pollution by pesticide residues is a major environmental concern due to the extensive use of these substances in agriculture. The insecticide beta-cyfluthrin is a synthetic pyrethroid widely used in agricultural and other domestic activities. The aim of the present study was to assess the genotoxic effects of a sublethal exposure of the fish Bryconamericus iheringii (Characidae) to a commercial formulation of beta-cyfluthrin using the comet assay. Fish were exposed to sublethal concentrations (4.2 and 5.6 microg/L) of beta-cyfluthrin under static conditions during 24- and 48-h exposure periods. Fish in tap water were used as negative controls. Results obtained by the comet assay revealed genotoxic effects of the pyrethroid in the higher concentration and at the longer exposure period. The mean DNA damage index of fish exposed to 5.6 microg/L beta-cyfluthrin for 48 h was significantly higher (145.9 +/- 51.8) than in the control group (69.3 +/- 39.5). These findings indicate that native fish species might be at risk for genotoxic damage in waters contaminated with beta-cyfluthrin.     

Genotoxic and antioxidant activities of ethoxyquin salts evaluated by the comet assay

Four newly synthesized salts of ethoxyquin (EQ: 1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline), an antioxidant used in animal feeds, were evaluated with the use of the comet assay performed on human lymphocytes: ethoxyquin ascorbate, ethoxyquin hexanoate, ethoxyquin salicylate and ethoxyquin salt of Trolox C (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid). In the study the abilities of these compounds to cause DNA fragmentation and to protect against H2O2-induced DNA damage were analysed. The obtained results were compared with those noted earlier for EQ. After EQ salts treatments (1-25 microM) the genotoxic effects were observed, but the genotoxic potentials of the compounds studied were lower than that of EQ. On the other hand, EQ salts, similarly to EQ, effectively protected the cells from oxidative effect of H2O2. EQ hexanoate was the most effective and its antioxidant activity was even slightly higher than that of EQ. We suggest that it is worth further detailed studies to estimate its usefulness as a preservative.     

Microbial cleavage of various organophosphorus insecticides.

Bacteria able to utilize Aspon, Azodrin, Dasanit, diazinon, malathion, Orthene, parathion, Trithion, dimethoate, Dylox, methyl parathion, and Vapona as sole phosphorus sources were isolated from soil and sewage. Individual isolates used from 3 to 10 of these insecticides as sole phosphorus sources. The extent of growth of two Pseudomonas strains in media containing diazinon and malathion was in the range expected from the amount of insecticide supplied, and their proliferation resulted in disappearance of the chemical. Resting cells of the pseudomonads derived from cultures grown on diazinon or malathion but not orthophosphate caused extensive destruction of these two organophosphates in the presence or absence of chloramphenicol. Extracts of the two bacteria derived from organophosphate-grown cultures catalyzed the disappearance of Aspon, Azodrin, Dasanit, diazinon, malathion, Orthene, parathion, and Trithion but not dimethoate, Dylox, methyl parathion, and Vapona. Results from gas chromatographic analysis suggested that the extracts formed dimethyl phosphate from azodrin, dimethyl phosphorodithioate from malathion, diethyl phosphorodithioate from Trithion, and diethyl phosphorothioate from Dasanit, diazinon, and parathion. Dimethyl phosphate, dimethyl phosphorothioate , dimethyl phosphorodithioate, diethyl phosphate, and diethyl phosphorothioate were not used by the pseudomonads as sole phosphorus sources.     

Genotoxicity of three pesticides used in Costa Rican banana plantations

The in vitro genotoxicity of imazalil and thiabendazole fungicides and the insecticide chlorpyrifos, compounds used in Costa Rican banana plantations, was evaluated with the single-cell gel electrophoresis technique (comet assay). The comet assay is a simple, rapid and low cost technique for quantification of DNA damage. This assay detects DNA single-strand breaks and alkali-labile sites in individual cells. The effects were analyzed by using human lymphocytes exposed to doses of 0, 25, 50, 75 and 100 microg/ml of each pesticide for 30 min at 37 degrees C. The cells were embedded in agarose, lysed, subjected to alkaline electrophoresis (pH >13) for 20 min at 25V, neutralized and dehydrated to be stained with a fluorescent dye and later comets visualization with the epifluorescence microscope. Chlorpyrifos and imazalil induced significant DNA damage in a dose-dependent manner. Chlorpyrifos was the major inductor of DNA breaks. These results indicate that both are genotoxic compounds in vitro. Thiabendazole fungicide did not induced DNA damage using the comet assay for all concentrations tested.     

Microbial cleavage of various organophosphorus insecticides.

Bacteria able to utilize Aspon, Azodrin, Dasanit, diazinon, malathion, Orthene, parathion, Trithion, dimethoate, Dylox, methyl parathion, and Vapona as sole phosphorus sources were isolated from soil and sewage. Individual isolates used from 3 to 10 of these insecticides as sole phosphorus sources. The extent of growth of two Pseudomonas strains in media containing diazinon and malathion was in the range expected from the amount of insecticide supplied, and their proliferation resulted in disappearance of the chemical. Resting cells of the pseudomonads derived from cultures grown on diazinon or malathion but not orthophosphate caused extensive destruction of these two organophosphates in the presence or absence of chloramphenicol. Extracts of the two bacteria derived from organophosphate-grown cultures catalyzed the disappearance of Aspon, Azodrin, Dasanit, diazinon, malathion, Orthene, parathion, and Trithion but not dimethoate, Dylox, methyl parathion, and Vapona. Results from gas chromatographic analysis suggested that the extracts formed dimethyl phosphate from azodrin, dimethyl phosphorodithioate from malathion, diethyl phosphorodithioate from Trithion, and diethyl phosphorothioate from Dasanit, diazinon, and parathion. Dimethyl phosphate, dimethyl phosphorothioate , dimethyl phosphorodithioate, diethyl phosphate, and diethyl phosphorothioate were not used by the pseudomonads as sole phosphorus sources.     

Application of Euglena gracilis cells to comet assay: evaluation of DNA damage and repair

Alkaline single-cell gel electrophoresis (comet assay) enables sensitive detection of DNA damage in eukaryotic cells induced by genotoxic agents. We performed a comet assay of unicellular green alga Euglena gracilis that was exposed to genotoxic chemicals, 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), benzo[a]pyrene (BAP), mitomycin C (MMC) and actinomycin D (AMD). Tail length and tail moment in migrated DNA were measured as indications of DNA damage. MNNG and BAP were found to cause concentration-dependent increases in DNA damage. The responses were more sensitive than those of human lymphocytes under the same treatment conditions. MMC and AMD showed no positive response, as reported elsewhere. The comet assays performed at specified times after treatment revealed that the DNA damaged by MNNG and gamma-ray irradiation was repaired during the initial 1h. The results clearly show that the comet assay is useful for evaluating chemically-induced DNA damage and repair in E. gracilis. Given the ease of culturing and handling E. gracilis as well as its sensitivity, the comet assay of this alga would undoubtedly prove to be a useful tool for testing the genotoxicity of chemicals and monitoring of environmental pollution.     

Genotoxicity of the volatile anaesthetic desflurane in human lymphocytes in vitro, established by comet assay

The aim of the present study was to estimate the genotoxicity of desflurane, applied as a volatile anaesthetic. The potential genotoxicity was determined by the comet assay as the extent of DNA fragmentation in human peripheral blood lymphocytes in vitro. The comet assay detects DNA strand breaks induced directly by genotoxic agents as well as DNA fragmentation due to cell death. Another anaesthetic, halothane, already proved to be a genotoxic agent, was used as a positive control. Both analysed drugs were capable of increasing DNA migration in a dose-dependent manner under experimental conditions applied. The results of the study demonstrated that the genotoxicity of desflurane was comparable with that of halothane. However, considering the pharmacodynamics of both drugs, the genotoxic activity of desflurane may be connected with a less harmful effect on the exposed patients or medical staff.