Evaluating the broad-spectrum efficacy of the acetylcholinesterase oximes reactivators MMB4 DMS, HLö-7 DMS, and 2-PAM Cl against phorate oxon, sarin, and VX in the Hartley guinea pig.

Organophosphorus (OP) compounds, including pesticides and chemical warfare nerve agents (CWNA), are threats to the general population as possible weapons of terrorism or by accidental exposure whether through inadvertent release from manufacturing facilities or during transport. To mitigate the toxicities posed by these threats, a therapeutic regimen that is quick-acting and efficacious against a broad spectrum of OPs is highly desired. The work described herein sought to assess the protective ratio (PR), median effective doses (ED50), and therapeutic index (TI = oxime 24-h LD50/oxime ED50) of MMB4 DMS, HLö-7 DMS, and 2-PAM Cl against the OPs sarin (GB), VX, and phorate-oxon (PHO). All OPs are representative of the broader classes of G and V chemical warfare nerve agents and persistent pesticides. MMB4 DMS and HLö-7 DMS were previously identified as comparative efficacy leads warranting further evaluations. 2-PAM Cl is the U.S. FDA-approved standard-of-care oxime therapy for OP intoxication. Briefly, PRs were determined in male guinea pigs by varying the subcutaneously (SC) delivered OP dose followed then by therapy with fixed levels of the oxime and atropine (0.4 mg/kg; administered intramuscularly [IM]). ED50s were determined using a similar approach except the OP dose was held constant at twice the median lethal dose (2 × LD50) while the oxime treatment levels were varied. The ED50 information was then used to calculate the TI for each OP/oxime combination. Both MMB4 DMS and HLö-7 DMS provided significant protection, i.e., higher PR against GB, VX, and PHO when compared to atropine controls, but significance was not readily demonstrated across the board when compared against 2-PAM Cl. The ED50 values of MMB4 DMS was consistently lower than that of the other oximes against all three OPs. Furthermore, based on those ED50s, the TI trend of the various oximes against both GB and VX was MMB4 DMS > HLö-7 DMS > 2-PAM Cl, while against PHO, MMB4 DMS > 2-PAM Cl > HLö-7 DMS.

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.

Evaluation of absorbent materials for use as ad hoc dry decontaminants during mass casualty incidents as part of the UK's Initial Operational Response (IOR).

The UK's Initial Operational Response (IOR) is a revised process for the medical management of mass casualties potentially contaminated with hazardous materials. A critical element of the IOR is the introduction of immediate, on-scene disrobing and decontamination of casualties to limit the adverse health effects of exposure. Ad hoc cleansing of the skin with dry absorbent materials has previously been identified as a potential means of facilitating emergency decontamination. The purpose of this study was to evaluate the in vitro oil and water absorbency of a range of materials commonly found in the domestic and clinical environments and to determine the effectiveness of a small, but representative selection of such materials in skin decontamination, using an established ex vivo model. Five contaminants were used in the study: methyl salicylate, parathion, diethyl malonate, phorate and potassium cyanide. In vitro measurements of water and oil absorbency did not correlate with ex vivo measurements of skin decontamination. When measured ex vivo, dry decontamination was consistently more effective than a standard wet decontamination method ("rinse-wipe-rinse") for removing liquid contaminants. However, dry decontamination was ineffective against particulate contamination. Collectively, these data confirm that absorbent materials such as wound dressings and tissue paper provide an effective, generic capability for emergency removal of liquid contaminants from the skin surface, but that wet decontamination should be used for non-liquid contaminants.

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.

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).

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.

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.

Preferential binding of insecticide phorate with sub-domain IIA of human serum albumin induces protein damage and its toxicological significance.

Phorate, an organophosphorus insecticide is known for its adverse effects on acetylcholinesterase, and other neuronal and pulmonary activities. Most likely, the toxicity of drugs/agrochemicals is modulated through cellular distribution bound to plasma proteins. Therefore, the in vitro interaction of phorate with human serum albumin (HSA) has been investigated, using sensitive techniques like fluorescence spectroscopy and circular dichroism, to ascertain its binding mechanism and toxicological implications. Fluorescence studies revealed the quenching constant (Ksv) as 2.5 × 104 M⁻¹ and binding affinity (Ka) as 2.96 × 104 M⁻¹ (r² = 0.99), with a primary binding site of phorate at sub-domain IIA of HSA. Circular dichroism (CD) data demonstrated a noticeable reduction in secondary structure (α-helical content) of phorate treated HSA. Albumin treated with 200-1000 µM phorate released significant amounts of acid soluble amino and carbonyl groups, whereas higher concentrations resulted in protein fragmentation. It is postulated that the 1'-O and 3-O alkyl groups of phorate have a role in binding with electrophilic centers of Trp 214, and Arg 218/Lys 195, respectively. Moreover, the significant ultrastructural changes, reactive oxygen species (ROS) generation, mitochondrial damage and cell death in phorate treated cultured human amnion epithelial (WISH) cells, elucidated phorate induced cellular toxicity.

Hypersalinity acclimation increases the toxicity of the insecticide phorate in coho salmon (Oncorhynchus kisutch).

Previous studies in euryhaline fish have shown that acclimation to hypersaline environments enhances the toxicity of thioether organophosphate and carbamate pesticides. To better understand the potential mechanism of enhanced toxicity, the effects of the organophosphate insecticide phorate were evaluated in coho salmon (Oncorhynchus kisutch) maintained in freshwater (<0.5 g/L salinity) and 32 g/L salinity. The observed 96-h LC50 in freshwater fish (67.34 ± 3.41 µg/L) was significantly reduced to 2.07 ± 0.16 µg/L in hypersaline-acclimated fish. Because organophosphates often require bioactivation to elicit toxicity through acetylcholinesterase (AChE) inhibition, the in vitro biotransformation of phorate was evaluated in coho salmon maintained in different salinities in liver, gills, and olfactory tissues. Phorate sulfoxide was the predominant metabolite in each tissue but rates of formation diminished in a salinity-dependent manner. In contrast, formation of phorate-oxon (gill; olfactory tissues), phorate sulfone (liver), and phorate-oxon sulfoxide (liver; olfactory tissues) was significantly enhanced in fish acclimated to higher salinities. From previous studies, it was expected that phorate and phorate sulfoxide would be less potent AChE inhibitors than phorate-oxon, with phorate-oxon sulfoxide being the most potent of the compounds tested. This trend was confirmed in this study. In summary, these results suggest that differential expression and/or catalytic activities of Phase I enzymes may be involved to enhance phorate oxidative metabolism and subsequent toxicity of phorate to coho salmon under hypersaline conditions. The outcome may be enhanced fish susceptibility to anticholineterase oxon sulfoxides.

Application of comet assay in the study of DNA damage and recovery in rohu (Labeo rohita) fingerlings after an exposure to phorate, an organophosphate pesticide.

Labeo rohita (rohu) fingerlings were exposed to different concentrations (0.001, 0.002 and 0.01 ppm) of phorate, an organophosphate pesticide; samplings were done at 24, 48, 72 and 96 h. The study was carried out to evaluate tissue specific genotoxic effects produced by phorate, on three different tissue systems and to assess DNA repair response in fish. Results of tissue specific DNA damage experiments showed low baseline damage in blood cells followed by gill and liver cells in control individuals whereas more DNA breaks were found in liver followed by gill and blood cells of treated individuals. Concentrations-dependent DNA damage showed a strong, linear and positive relationship (r(2) = >0.7) in all three tissues. Clear time-related increase in DNA damage was observed for all tissues exposed to all concentrations except in liver cells at 0.01 ppm, where the DNA damage declined significantly after 72 h. For the assessment of DNA repair response, fingerlings were first exposed to 0.01 ppm of phorate for 72 h and then transferred to pesticide free water. Tissue chosen for the repair experiment was liver. Samplings were done at 0, 3, 6, 12 and 24 h after the release of 72 h pesticide treated fishes into pesticide free water. Fishes showed a reduction in DNA breaks from 3 h onwards in pesticide free water and at 24 h returned to control level damage. The results indicate that phorate is a potential genotoxicant, comet assay can be used in DNA damage and repair analysis, response to pollutants in multicellular animals is often tissue specific.

Phorate-induced enzymological alterations in mouse olfactory bulb.

The organophosphate pesticide, phorate, is an extremely hazardous insecticide. Not much experimental study is available on effects of phorate on different brain areas. We report in this study the alterations induced by phorate on enzyme profile of mouse olfactory bulb. Olfactory bulb, the first processing centre after the sensory cells in the olfactory pathway, has connections with the other higher centres of the brain like hippocampus and hypothalamus. Phorate was administered orally in the diet at the doses of 1.0 mg and 1.5 mg/kg body weight to adult albino mice. After 32 weeks of exposure animals were sacrificed and cryosections were processed for acetylcholinesterase and butyrylcholinesterase (AChE and BChE, respectively) enzyme localization. Significant reduction occurs in AChE and BChE activity at higher dose level, whereas reduced BChE activity was found at both dose levels. Our results shows an obvious effect on cholinesterase enzyme profile of olfactory bulb of mice after systemic administration of low doses of phorate for long terms.

Fate and impact of pesticides applied to potato cultures: the Nicolet River basin.

The fate of cash-crop (potato) pesticides was monitored from the fields on which they were applied to the nearby streams. The investigation took place in the Nicolet River basin in the province of Quebec, Canada. The main pesticides under study were aldicarb, fenvalerate, metribuzin, and phorate. Aldicarb was never detected in any of the samples. The other pesticides were all detected in soils at low concentrations. Only fenvalerate and metribuzin were detected in tile drain. Metribuzin concentrations of up to 0.25 microgram/g were detected in the soil giving rise to a concentration of 1.3 micrograms/liter in tile drain and 47.1 micrograms/liter in surface runoff. Low concentrations of metribuzin up to 0.41 microgram/liter were detected in the nearby streams. The CREAMS model simulating pesticide movement in the fields overestimated metribuzin losses in the runoff at a concentration of 107 micrograms/liter. The subsurface EXPRES model using a PRZM time series adequately estimated a metribuzin field subsurface runoff concentration of 0.5 microgram/liter. According to the Canadian Water Quality Guideline for the protection of aquatic life, the concentrations of pesticides found in surface waters of this potato-growing region of Quebec do not have a potential to impact on the aquatic life in these systems.

Pesticide clastogenicity in Chinese hamster ovary cells.

Paraquat, alachlor, butachlor, phorate and monocrotophos, several of the most extensively used pesticides in Taiwan, were investigated for their clastogenicity using chromosome aberration (CAb) induction in Chinese hamster ovary (CHO) cells. Significance levels of the binomial trend analysis and binomial mutagenicity data test were two criteria for the summary judgement of the pesticide clastogenicity. Except for phorate, all pesticides tested were clastogenic to CHO cells in the absence of in vitro metabolic activation by S9. 5 microliters/ml rat-liver extract, S9, were used as the source of in vitro metabolic activation. 3 different outcomes were found after the addition of S9. Paraquat: significant decrease in induced CAbs. Monocrotophos: concomitant occurrence of decreased cytotoxicity and increased clastogenicity. Alachlor, butachlor and phorate: increased cytotoxicities with no sign of enhancement in clastogenicity.

Evaluation of Thimet 10-G for mutagenicity by 4 different genetic systems.

The insecticide Thimet 10-G was tested for mutagenic activity by 4 different genetic systems. It was unable to induce gene mutation in Salmonella, transfection inhibition in Mycobacterium, micronuclei formation in mice, and sister-chromatid exchange (SCE) in human lymphocytes were evaluated. It caused in mice an increase in the ratio of normochromatic to polychromatic erythrocytes and in human lymphocytes a decrease in mitotic index and delay in cell cycle. The results indicate that the insecticide is not mutagenic in the 4 test systems used at present.

Clinical effects and cholinesterase activity changes in workers exposed to Phorate (Thimet).

Phorate (Thimet), an aliphatic derivative of phosphorus is a highly toxic insecticide. In order to implement the safety measures, the clinical manifestations and cholinesterase (ChE) activity were evaluated before and after 2 weeks of exposure to this insecticide in 40 male formulators. The 2 week's exposure reveal signs and symptoms of toxicity in 60% of the formulators. Gastrointestinal symptoms and lowering of heart rate (bradycardia) were more prominent as compared to the neurological symptoms. A significant depression in plasma ChE activity was observed at the end of 1st week (55%) and 2nd week (71%) as compared to the respective pre-exposure values. A recovery up to 79% of the pre-exposure activity of this enzyme was noticed 10 days after cessation of the above exposure.