Biodegradation of phorate by bacterial strains in the presence of humic acid and metal ions.

Phorate is a systemic insecticide used to eradicate mites, insects, and nematodes. Extensive use of this organophosphate has engendered severe environmental concerns. The current research aimed to explore the kinetic pathways of phorate biodegradation in aqueous solutions. Two novel bacterial strains Pseudomonas aeruginosa strain PR1 (KP268772.1) and Pseudomonas sp. PR_02 (KP268773.1) were isolated, screened, and developed given their potential to degrade phorate. Mineralization of phorate was assayed with and without the addition of metal ions [Fe (II) and Cu (II)] and humic acid (HA). In 14 days, experiment both strains have consumed about 69%-94.5% (half-life from 3.58 to 6.02 days) of phorate. The observed biodegradation rate of phorate with Cu (II) in the system was 73% and 87%, with a half-life of 4.86 and 4.07 days for PR1 and PR2, respectively. The biodegradation of phorate using Fe(II) was 69% and 82%, with half-life periods 5.68 and 4.49 days. Meanwhile, incorporating HA, the phorate biodegradation was inhibited significantly, showing 71% and 85% degradation, with half-life periods of 6.02 and 5.02 days. The results indicated that both bacterial strains were able to mineralize phorate with PR2 > PR1. Summarizing, the inhibition in phorate biodegradation order under different conditions was as HA > Fe (II) > Cu (II). UV-visible measurements and gas chromatography-mass spectrometric assays indicated that the possible degradation pathway of phorate included ethoxy-phosphonothio-methanethiol S-mercaptomethyl-O,O-dihydrogen phosphorodithioate, diethyl-methylphosphonate, methane dithiol, ethanethiol, and phosphate, as the main metabolites identified. Therefore, it was concluded that the newly isolated Pseudomonas strains could be a potential candidates for biodegradation of phorate in a cost-effective, safe, and environmentally friendly alternative.

Application of Q-TOF-MS based metabonomics techniques to analyze the plasma metabolic profile changes on rats following death due to acute intoxication of phorate.

Organophosphorus pesticides (OPS) are widely used in the world, and many poisoning cases were caused by them. Phorate intoxication is especially common in China. However, there are currently few methods for discriminating phorate poisoning death from phorate exposure after death and interpretation of false-positive results due to the lack of effective biomarkers. In this study, we investigated the metabonomics of rat plasma at different dose levels of acute phorate intoxication using ultra-performance liquid chromatography quadrupole-time of flight mass spectrometry (UPLC-Q-TOF-MS) analysis. A total of 11 endogenous metabolites were significantly changed in the groups exposed to phorate at LD50 level and three times of LD50 (3LD50) level compared with the control group, which could be potential biomarkers of acute phorate intoxication. Plasma metabonomics analysis showed that diethylthiophosphate (DETP) could be a useful biomarker of acute phorate intoxication. The levels of uric acid, acylcarnitine, succinate, gluconic acid, and phosphatidylcholine (PC) (36:2) were increased, while pyruvate level was decreased in all groups exposed to phorate. The levels of ceramides (Cer) (d 18:0/16:0), palmitic acid, and lysophosphatidylcholine (lysoPC) (18:1) were only changed after 3LD50 dosage. The results of this study indicate that the dose-dependent relationship exists between metabolomic profile change and toxicities associated with apoptosis, fatty acid metabolism disorder, energy metabolism disorder especially tricarboxylic acid (TCA) cycle, as well as liver, kidney, and nervous system functions after acute exposure of phorate. This study shows that metabonomics is a useful tool in identifying biomarkers for the forensic toxicology study of phorate poisoning.

Pesticide Residues Identification by Optical Spectrum in the Time-Sequence of Enzyme Inhibitors Performed on Microfluidic Paper-Based Analytical Devices (µPADs).

Pesticides vary in the level of poisonousness, while a conventional rapid test card only provides a general "absence or not" solution, which cannot identify the various genera of pesticides. In order to solve this problem, we proposed a seven-layer paper-based microfluidic chip, integrating the enzyme acetylcholinesterase (AChE) and chromogenic reaction. It enables on-chip pesticide identification via a reflected light intensity spectrum in time-sequence according to the different reaction efficiencies of pesticide molecules and assures the optimum temperature for enzyme activity. After pretreatment of figures of reflected light intensity during the 15 min period, the figures mainly focused on the reflected light variations aroused by the enzyme inhibition assay, and thus, the linear discriminant analysis showed satisfying discrimination of imidacloprid (Y = -1.6525X - 139.7500), phorate (Y = -3.9689X - 483.0526), and avermectin (Y = -2.3617X - 28.3082). The correlation coefficients for these linearity curves were 0.9635, 0.8093, and 0.9094, respectively, with a 95% limit of agreement. Then, the avermectin class chemicals and real-world samples (i.e., lettuce and rice) were tested, which all showed feasible graphic results to distinguish all the chemicals. Therefore, it is feasible to distinguish the three tested kinds of pesticides by the changes in the reflected light spectrum in each min (15 min) via the proposed chip with a high level of automation and integration.

Bioremediation of organophosphorus pesticide phorate in soil by microbial consortia.

Microbial consortia isolated from aged phorate contaminated soil were used to degrade phorate. The consortia of three microorganisms (Brevibacterium frigoritolerans, Bacillus aerophilus and Pseudomonas fulva) could degrade phorate, and the highest phorate removal (between 97.65 and 98.31%) was found in soils inoculated with mixed cultures of all the three bacterial species. However, the mixed activity of any of two of these bacteria was lower than mixed consortia of all the three bacterial species. The highest degradation by individual mixed consortia of (B. frigoritolerans+B.aerophilus, B. aerophilus+P. fulva and B. frigoritolerans+P. fulva) appeared in soil between (92.28-94.09%, 95.45-97.15% and 94.08-97.42%, respectively). Therefore, inoculation of highly potential microbial consortia isolated from in situ contaminated soil could result in most effective bioremediation consortia for significantly relieving soils from phorate residues. This much high phorate remediation from phorate contaminated soils have never been reported earlier by mixed culture of native soil bacterial isolates.

Kinetic analysis of oxime-assisted reactivation of human, Guinea pig, and rat acetylcholinesterase inhibited by the organophosphorus pesticide metabolite phorate oxon (PHO).

Phorate is a highly toxic agricultural pesticide currently in use throughout the world. Like many other organophosphorus (OP) pesticides, the primary mechanism of the acute toxicity of phorate is acetylcholinesterase (AChE) inhibition mediated by its bioactivated oxon metabolite. AChE reactivation is a critical aspect in the treatment of acute OP intoxication. Unfortunately, very little is currently known about the capacity of various oximes to rescue phorate oxon (PHO)-inhibited AChE. To help fill this knowledge gap, we evaluated the kinetics of inhibition, reactivation, and aging of PHO using recombinant AChE derived from three species (rat, guinea pig and human) commonly utilized to study the toxicity of OP compounds and five oximes that are currently fielded (or have been deemed extremely promising) as anti-OP therapies by various nations around the globe: 2-PAM Cl, HI-6 DMS, obidoxime Cl2, MMB4-DMS, and HLö7 DMS. The inhibition rate constants (ki) for PHO were calculated for AChE derived from each species and found to be low (i.e., 4.8×103 to 1.4×104M-1min-1) compared to many other OPs. Obidoxime Cl2 was the most effective reactivator tested. The aging rate of PHO-inhibited AChE was very slow (limited aging was observed out to 48h) for all three species. CONCLUSIONS: (1) Obidoxime Cl2 was the most effective reactivator tested. (2) 2-PAM Cl, showed limited effectiveness in reactivating PHO-inhibited AChE, suggesting that it may have limited usefulness in the clinical management of acute PHO intoxication. (3) The therapeutic window for oxime administration following exposure to phorate (or PHO) is not limited by aging.

[Clinical effect of alanyl glutamine in the treatment of patients with gastrointestinal function obstacle caused by severe phorate poisoning].

Objective: To observe the therapeutic efficacy of alanyl glutamine injection on patients with gastrointestinal function obstacle caused by severe phorate poisoning. Methods: A total of 80 eligible patients with gastrointestinal function obstacle caused by severe phorate poisoning were randomly divided into the control group (n=40) and treatment group (n=40) . The control group was treated with the conventional therapy, which included forbidden diet, atropine, pralidoxime iodide, anti-inflammatory, albumin infusion, ω-3 fish oil fat emulsion, protection of organs function, blood perfusion, and Fat Emulsion, Amino Acids (17) and Glucose Injection. The treatment group was treated with alanyl glutamine injection plus the conventional therapy. To observe the time of recovering to normal of gastrointestinal function between the two groups, compared the AChE activity and changes of prealbumin, albumin and total protein of the two groups respectively. Furthermore, the total atropine dosage, the total pralidoxime iodide dosage and ICU stay time between the two groups were also compared. Results: The gastrointestinal function recovery time of patients in the treatment group was less than the control group, the difference was statistically significant (P<0.05) . From the third day of treatment, the serum cholinesterase activity of the treatment group was higher than the control group, the difference was statistically significant (P<0.05) . On the 5th day and 10th day of the treatment, the prealbumin, albumin and total protein of the treatment group were significantly higher than these indexes of the control group in the same period, the difference were statistically significant (P<0.05) . The total atropine dosage, the total pralidoxime iodide dosage and ICU stay time in the treatment group were lower than the control group, the difference were statistically significant (P<0.05) . Conclusion: Alanyl glutamine injection has a great therapeutic effect for gastrointestinal function obstacle patients caused by severe phorate poisoning.

Development of gold nanoparticles-based aptasensor for the colorimetric detection of organophosphorus pesticide phorate.

The present study reports a highly simple and rapid method for the detection of a widely used and extremely toxic organophosphorus pesticide, phorate. The detection employs a pesticide-specific aptamer as the recognition element and gold nanoparticles as the optical sensors. The aptamer, owing to its random coil structure, provides stability to the gold nanoparticles upon linking, thereby keeping the nanoparticles well dispersed. However, on the addition of the target pesticide, the aptamer acquires a rigid conformation resulting in the aggregation of the gold nanoparticles. Consequently, the color of the solution changes from red to blue and is easily observable with the naked eye. The proposed method was linear in the concentration range of 0.01 nM to 1.3 µm with the limit of detection as low as 0.01 nM. Moreover, the proposed assay selectively recognized phorate in the presence of other interfering substances and, thus, can be applied to real samples for the rapid and efficient screening of phorate.

QuEChERS method for the simultaneous quantification of phorate and its metabolites in porcine and chicken muscle and table eggs using ultra-high performance liquid chromatography with tandem mass spectrometry.

An analytical method to detect phorate and its metabolites, including phorate sulfone, phorate sulfoxide, phoratoxon, phoratoxon sulfone, and phoratoxon sulfoxide, in porcine and chicken muscles and table eggs was developed and validated. Extraction was performed using a quick, easy, cheap, effective, rugged, and safe method and analysis was conducted using ultra-high performance liquid chromatography-tandem mass spectrometry. Matrix-matched calibrations were linear over the tested concentrations, with determination coefficient ≥ 0.995 for all tested analytes in the different matrices. The limits of detection and quantification were 0.001 and 0.004 mg/kg, respectively. The calculated recovery rates at three fortification levels were satisfactory, with values between 74.22 and 119.89% and relative standard deviations < 10%. The method was applied successfully to commercial samples collected from locations throughout the Korean Peninsula, and none of them showed any traces of the tested analytes. Overall, the developed method is simple and versatile, and can be used for monitoring phorate and its metabolites in animal products rich in protein and fat.

Dissipation behavior of phorate and its toxic metabolites in the sandy clay loam soil of a tropical sugarcane ecosystem using a single-step sample preparation method and GC-MS.

The dissipation of phorate in the sandy clay loam soil of tropical sugarcane ecosystem was studied by employing a single-step sample preparation method and gas chromatography with mass spectrometry. The limit of quantification of the method was 0.01 µg/g. The recoveries of phorate, phorate sulfoxide, phorate sulfone, and phorate oxon were in the range 94.00-98.46% with relative standard deviations of 1.51-3.56% at three levels of fortification between 0.01 and 0.1 µg/g. The Half-life of phorate and the total residues, which include phorate, phorate sulfoxide and phorate sulfone, was 5.5 and 19.8 days, respectively at the recommended dose of insecticide. Phorate rapidly oxidized into its sulfoxide metabolite in the sandy clay loam soil. Phorate sulfoxide alone accounted for more than 20% of the total residues within 2 h post-application and it was more than 50% on the fifth day after treatment irrespective of the doses applied. Phorate sulfoxide and phorate sulfone reached below the detectable level on 105 and 135 days after treatment, respectively as against 45 days after treatment for phorate residues at the recommended dose. Thus, the reasonably prolonged efficacy of phorate against soil pests may be attributed to longer persistence of its more toxic sulfoxide and sulfone metabolites.

Isolation and evaluation of potent Pseudomonas species for bioremediation of phorate in amended soil.

Use of phorate as a broad spectrum pesticide in agricultural crops is finding disfavor due to persistence of both the principal compound as well as its toxic residues in soil. Three phorate utilizing bacterial species (Pseudomonas sp. strain Imbl 4.3, Pseudomonas sp. strain Imbl 5.1, Pseudomonas sp. strain Imbl 5.2) were isolated from field soils. Comparative phorate degradation analysis of these species in liquid cultures identified Pseudomonas sp. strain Imbl 5.1 to cause complete metabolization of phorate during seven days as compared to the other two species in 13 days. In soils amended with phorate at different levels (100, 200, 300 mg kg(-1) soil), Pseudomonas sp. strain Imbl 5.1 resulted in active metabolization of phorate by between 94.66% and 95.62% establishing the same to be a potent bacterium for significantly relieving soil from phorate residues. Metabolization of phorate to these phorate residues did not follow the first order kinetics. This study proves that Pseudomonas sp. strain Imbl 5.1 has huge potential for active bioremediation of phorate both in liquid cultures and agricultural soils.

Brevibacterium frigoritolerans as a Novel Organism for the Bioremediation of Phorate.

Phorate, an organophosphorus insecticide, has been found effective for the control of various insect pests. However, it is an extremely hazardous insecticide and causes a potential threat to ecosystem. Bioremediation is a promising approach to degrade the pesticide from the soil. The screening of soil from sugarcane fields resulted in identification of Brevibacterium frigoritolerans, a microorganism with potential for phorate bioremediation was determined. B. frigoritolerans strain Imbl 2.1 resulted in the active metabolization of phorate by between 89.81% and 92.32% from soils amended with phorate at different levels (100, 200, 300 mg kg(-1) soil). But in case of control soil, 33.76%-40.92% degradation were observed. Among metabolites, sulfone was found as the main metabolite followed by sulfoxide. Total phorate residues were not found to follow the first order kinetics. This demonstrated that B. frigoritolerans has potential for bioremediation of phorate both in liquid cultures and agricultural soils.

Determination of Organothiophosphate Insecticides in Environmental Water Samples by a Very Simple and Sensitive Spectrofluorimetric Method.

A simple, rapid and sensitive spectrofluorimetric method was developed for the determination of di-syston, ethion and phorate in environmental water samples. The procedure is based on the oxidation of these pesticides with cerium (IV) to produce cerium (III), and its fluorescence was monitored at 368 ± 3 nm after excitation at 257 ± 3 nm. The variables effecting oxidation of each pesticide were studied and optimized. Under the experimental conditions used, the calibration graphs were linear over the range 0.2-15, 0.1-13, 0.1-13 ng mL(-1) for di-syston, ethion and phorate, respectively. The limit of detection and quantification were in the range 0.034-0.096 and 0.112-0.316 ng mL(-1), respectively. Intra- and inter-day assay precisions, expressed as the relative standard deviation (RSD), were lower than 5.2 % and 6.7 %, respectively. Good recoveries in the range 86 %-108 % were obtained for spiked water samples. The proposed method was applied to the determination of studied pesticides in environmental water samples.

Metabonomics evaluation of urine from rats administered with phorate under long-term and low-level exposure by ultra-performance liquid chromatography-mass spectrometry.

The purpose of this study was to investigate the toxic effect of long-term and low-level exposure to phorate using a metabonomics approach based on ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). Male Wistar rats were given phorate daily in drinking water at low doses of 0.05, 0.15 or 0.45 mg kg⁻¹ body weight (BW) for 24 weeks consecutively. Rats in the control group were given an equivalent volume of drinking water. Compared with the control group, serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TBIL), urea nitrogen (BUN) and creatinine (CR) were increased in the middle- and high-dose groups whereas albumin (ALB) and cholinesterase (CHE) were decreased. Urine metabonomics profiles were analyzed by UPLC-MS. Compared with the control group, 12 metabolites were significantly changed in phorate-treated groups. In the negative mode, metabolite intensities of uric acid, suberic acid and citric acid were significantly decreased in the middle- and high-dose groups, whereas indoxyl sulfic acid (indican) and cholic acid were increased. In the positive mode, uric acid, creatinine, kynurenic acid and xanthurenic acid were significantly decreased in the middle- and high-dose groups, but 7-methylguanine (N7G) was increased. In both negative and positive modes, diethylthiophosphate (DETP) was significantly increased, which was considered as a biomarker of exposure to phorate. In conclusion, long-term and low-level exposure to phorate can cause disturbances in energy-related metabolism, liver and kidney function, the antioxidant system, and DNA damage. Moreover, more information can be provided on the evaluation of toxicity of phorate using metabonomics combined with clinical chemistry.

Assessment of the genotoxic effects of organophosphorus insecticides phorate and trichlorfon in human lymphocytes.

In vitro genotoxic effects of organophosphorus insecticides Phorate (PHR) and Trichlorfon (TCF) were investigated using four genotoxicity endpoints. Different concentration ranges between 0.25-2.00 µg mL(-1) of PHR and 2.34-37.50 µg mL(-1) of TCF were applied to lymphocytes. PHR and TCF significantly increased the frequency of chromosomal aberrations (except 2.34 µg mL(-1) for TCF) and sister chromatid exchanges at all treatment times and concentrations. Most of the used concentrations induced a significant increase in the frequency of micronuclei. Furthermore, PHR and TCF significantly decreased the mitotic index at the higher concentrations after 24- and 48-h treatments. In the comet assay, PHR and TCF significantly increased the comet tail at all concentrations. However, the comet tail intensity was significantly increased at only the highest concentration of PHR and at all concentrations of TCF. According to these results, PHR and TCF possess clastogenic, mutagenic, and DNA damaging effects in human lymphocytes in vitro.

Bioelectrochemical platform with human monooxygenases: FMO1 and CYP3A4 tandem reactions with phorate.

It is highly advantageous to devise an in vitro platform that can predict the complexity of an in vivo system. The first step of this process is the identification of a xenobiotic whose monooxygenation is carried out by two sequential enzymatic reactions. Pesticides are a good model for this type of tandem reactions since in specific cases they are initially metabolised by human flavin-containing monooxygenase 1 (hFMO1), followed by cytochrome P450 (CYP). To assess the feasibility of such an in vitro platform, hFMO1 is immobilised on glassy carbon electrodes modified with graphene oxide (GO) and cationic surfactant didecyldimethylammonium bromide (DDAB). UV-vis, contact angle and AFM measurements support the effective decoration of the GO sheets by DDAB which appear as 3 nm thick structures. hFMO1 activity on the bioelectrode versus three pesticides; fenthion, methiocarb and phorate, lead to the expected sulfoxide products with KM values of 29.5 ± 5.1, 38.4 ± 7.5, 29.6 ± 4.1 µM, respectively. Moreover, phorate is subsequently tested in a tandem system with hFMO1 and CYP3A4 resulting in both phorate sulfoxide as well as phoratoxon sulfoxide. The data demonstrate the feasibility of using bioelectrochemical platforms to mimic the complex metabolic reactions of xenobiotics within the human body.

Environmental fate and metabolic transformation of two non-ionic pesticides in soil: Effect of biochar, moisture, and soil sterilization.

Soil moisture, organic matter, and soil microbes are the key considering factors that control the persistence, degradation, and transformation of applied pesticides under varied soil conditions. In this study, underlying influence of these factors was assessed through the fates and metabolic transformation of two non-ionic pesticides (e.g., Phorate and Terbufos) in soils. Concisely, two distinct experiments including a customized batch equilibrium (sorption study), and a lab incubation trial (degradation study) were performed, following the OECD guidelines. As per study findings, biochar (BC) amendment was found to be the most influential factors during sorption study, particularly, 1% BC amendment contributed to achieve the best results. In addition, the non-linearity of sorption isotherm (1/n < 1.0) was revealed through Freundlich isotherm, indicating the strong adsorption of studied pesticides onto the soils. On the other hand, during degradation study, soil moisture initiates the enhanced degradation of parent pesticides and subsequent metabolism. In the presence of 40% water holding capacity (WHC), 1% BC amendment enhances the metabolic transformation, while H2O2 treatment could hinder the process. Additionally, the half-life degradation (t1/2) of phorate and terbufos was controlled by biochar amendment, moisture, and soil sterilization, respectively. Finally, BC can accelerate the metabolic transformation, whereas, phorate underwent a metabolic change into sulfoxide and sulfone while terbufos turned into solely sulfoxide. This pioneering study gathered crucial data for understanding the persistence and metabolic transition of non-ionic pesticides in soils and their patterns of degradation.

Highly Sensitive Fluorescence Detection of Three Organophosphorus Pesticides Based on Highly Bright DNA-Templated Silver Nanoclusters.

It is still challenging to achieve simultaneous and sensitive detection of multiple organophosphorus pesticides (OPs). Herein, we optimized the ssDNA templates for the synthesis of silver nanoclusters (Ag NCs). For the first time, we found that the fluorescence intensity of T base-extended DNA-templated Ag NCs was over three times higher than the original C-riched DNA-templated Ag NCs. Moreover, a "turn-off" fluorescence sensor based on the brightest DNA-Ag NCs was constructed for the sensitive detection of dimethoate, ethion and phorate. Under strong alkaline conditions, the P-S bonds in three pesticides were broken, and the corresponding hydrolysates were obtained. The sulfhydryl groups in the hydrolyzed products formed Ag-S bonds with the silver atoms on the surface of Ag NCs, which resulted in the aggregation of Ag NCs, following the fluorescence quenching. The fluorescence sensor showed that the linear ranges were 0.1-4 ng/mL for dimethoate with a limit of detection (LOD) of 0.05 ng/mL, 0.3-2 µg/mL for ethion with a LOD of 30 ng/mL, and 0.03-0.25 µg/mL for phorate with a LOD of 3 ng/mL. Moreover, the developed method was successfully applied to the detection of dimethoate, ethion and phorate in lake water samples, indicating a potential application in OP detection.

Phorate-induced oxidative stress, DNA damage and transcriptional activation of p53 and caspase genes in male Wistar rats.

Male Wistar rats exposed to a systemic organophosphorus insecticide, phorate [O,O-diethyl S-[(ethylthio) methyl] phosphorothioate] at varying oral doses of 0.046, 0.092 or 0.184mg phorate/kg bw for 14days, exhibited substantial oxidative stress, cellular DNA damage and activation of apoptosis-related p53, caspase 3 and 9 genes. The histopathological changes including the pyknotic nuclei, inflammatory leukocyte infiltrations, renal necrosis, and cardiac myofiber degeneration were observed in the liver, kidney and heart tissues. Biochemical analysis of catalase and glutathione revealed significantly lesser activities of antioxidative enzymes and lipid peroxidation in tissues of phorate exposed rats. Furthermore, generation of intracellular reactive oxygen species and reduced mitochondrial membrane potential in bone marrow cells confirmed phorate-induced oxidative stress. Significant DNA damage was measured through comet assay in terms of the Olive tail moment in bone marrow cells of treated animals as compared to control. Cell cycle analysis also demonstrated the G(2)/M arrest and appearance of a distinctive SubG(1) peak, which signified induction of apoptosis. Up-regulation of tumor suppressor p53 and caspase 3 and 9 genes, determined by quantitative real-time PCR and enzyme-linked immunosorbent assay, elucidated the activation of intrinsic apoptotic pathways in response to cellular stress. Overall, the results suggest that phorate induces genetic alterations and cellular toxicity, which can adversely affect the normal cellular functioning in rats.

Cytotoxic and necrotic responses in human amniotic epithelial (WISH) cells exposed to organophosphate insecticide phorate.

The in vitro interaction of the organophosphorous insecticide (OPs) phorate with calf thymus DNA (ctDNA), and its potential to cause changes in cell cycle, membrane damage, and cytotoxicity leading to cell death (necrosis) was investigated in human amnion epithelial (WISH) cells. Fluorescence quenching revealed high binding affinity (K(a)=5.62×10(4)M(-1)) of phorate to ctDNA. Molecular modeling of the phorate-ctDNA interaction suggested the binding of phorate at AT rich regions on minor groove of DNA. The interaction ensued alkylation of the N-6, N-7 of adenine and C-4 carbonyl oxygen of thymine. Binding of phorate was stronger in the presence of the transition metal ion copper II (Cu(2+)), and has accentuated the destabilization of the DNA secondary structure. A discernable change in the voltammetric E(1/2) (E(0')) with lesser cathodic (i(pc)) and anodic (i(pa)) peak currents confirmed the formation of phorate-DNA and phorate-DNA-Cu (II) association complexes. Furthermore, the MTT and NRU assays demonstrated substantial phorate cytotoxicity due to loss of mitochondrial and lysosomal membrane integrity, and reduction in mitochondrial membrane potential (ΔΨm) of treated WISH cells. Cell cycle analysis of WISH cells treated with 1000µM phorate exhibited 13.7-fold (p<0.01) augmentation in the sub-G(1) peak. Annexin V-PE and 7-ADD staining of phorate treated cells reaffirmed the development of late apoptotic or necrotic cell population in a concentration dependent manner. Thus, this study demonstrated the phorate induced DNA structural alterations and cellular damage in cultured human cells.

Variation in effects of four OP insecticides on photosynthetic pigment fluorescence of Chlorella vulgaris Beij.

Effects of the insecticides quinalphos, chlorfenvinphos, dimethoate and phorate on photosystem activity of Chlorella vulgaris were investigated by different chlorophyll fluorescence measurements. Exposure to each of the insecticides increased the proportion of inactivated PS II reaction center. Quinalphos and chlorfenvinphos caused OJIP fluorescence reduction at all levels by decreasing the proportion of Q(A)-reducing PS II reaction centers (RCs). The other two insecticides affected OJIP fluorescence rise by hindering the electron transport beyond Q(A). Insecticide treatment resulted in decrease of the density of active RC and performance indices (PI) by enhanced dissipated energy flux per active RC. Antenna size was severely minimized by quinalphos and chlorfenvinphos treatment whereas other two insecticides had no such effect. Each insecticide treatment caused increase of photosystem antenna/core and PS II/PS I fluorescence ratios. Quinalphos and chlorfenvinphos affected the donor sides of photosystems whereas dimethoate and phorate inhibited electron transfer beyond Q(A) (acceptor side).