The effect of two bromfenvinphos impurities: BDCEE and ß-ketophosphonate on oxidative stress induction, acetylcholinesterase activity, and viability of human red blood cells.

Numerous research works have shown that synthesis of pesticides leads to the formation of impurities that may substantially enhance pesticide toxicity. In this study, the effect of manufacturing impurities of pesticide bromfenvinphos (BFVF) such as 1-bromo-2-(2,4-dichlorophenyl)-2-ethoxy ethene (BDCEE) and diethyl [2-(2,4-dichlorophenyl)-2-oxo-ethyl] phosphonate (ß-ketophosphonate) on human erythrocytes, being significantly exposed to xenobiotics has been studied. The cells were treated with the compounds studied in the concentrations ranging from 0.1 µM to 250 µM for 4 h. In order to assess the effect of BDCEE and ß-ketophosphonate on red blood cells hemolytic changes, changes in cell size (FSC parameter) and oxidation of hemoglobin were studied. Moreover, alterations in reactive oxygen species (ROS) formation, reduced glutathione (GSH) level and acetylcholinesterase (AChE) activity were determined. BDCEE induced an increase in ROS level and caused strong oxidation of hemoglobin as well as a slight change in erythrocytes size and hemolysis, while it did not change GSH level and AChE activity. ß-ketophosphonate has not been shown to affect most parameters studied, but it strongly reduced AChE activity. Because changes in the parameters examined were noted at low concentrations of BFVF impurities (5-250 µM), those substances should not negatively affect on red blood cells of humans environmentally exposed to this pesticide.

Bromfenvinphos induced suicidal death of human erythrocytes.

The organophosphorus pesticide bromfenvinphos ((E,Z)-O,O-diethyl-O-[1-(2,4-dichlorophenyl)-2-bromovinyl] phosphate) has been shown to decrease hematocrit and hemoglobin levels in blood presumably by triggering oxidative stress of erythrocytes. Oxidative stress is known to activate erythrocytic Ca(2+) permeable unselective cation channels leading to Ca(2+) entry and increase of cytosolic Ca(2+) activity ([Ca(2+)]i), which in turn triggers eryptosis, the suicidal death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. The present study explored, whether and how bromfenvinphos induces eryptosis. To this end, phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, hemolysis from hemoglobin release, [Ca(2+)]i from Fluo3-fluorescence, and ROS formation from DCFDA dependent fluorescence. As a result, a 48hour exposure of human erythrocytes to bromfenvinphos (≥100µM) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter, significantly increased Fluo3-fluorescence, and significantly increased DCFDA fluorescence. The effect of bromfenvinphos on annexin-V-binding and forward scatter was significantly blunted, but not abolished by removal of extracellular Ca(2+). In conclusion, bromfenvinphos triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to stimulation of ROS formation and Ca(2+) entry.

Oxidative stress in human erythrocytes treated with bromfenvinphos and its impurities.

Bromfenvinphos (BFVF) is an organophosphorus (OP) pesticide which was widely used in agriculture and veterinary practice. During synthesis of this insecticide five main impurities are formed: dihydro-bromfenvinphos, dibromo-bromfenvinphos, 2,4-dichlorophenacyl bromide, 2,4-dichlorophenacylidene bromide and 2,4-dichlorophenacylidyne bromide, which can be present in technical grade bromfenvinphos in amounts from 0.1 to 4%. The aim of this study was to examine the influence of bromfenvinphos and its manufacturing impurities on parameters of oxidative stress, the activity of antioxidative enzymes and the level of reduced glutathione. Human erythrocytes were incubated with bromfenvinphos and its impurities in the concentrations range from 0.5 to 500 µM for 1 h. This study indicated that 2,4-dichlorophenacyl derivatives more strongly oxidized analyzed parameters in human erythrocytes than bromfenvinphos. Investigated compounds caused an increase in lipid peroxidation and oxidation of fluorescent probe DCFH2 - the strongest pro-oxidative changes were provoked by 2,4-dichlorophenacyl bromide. None of the compounds studied in the concentrations from 0.5 to 500 µM changed the activity of SOD and only 2,4-dichlorophenacyl decreased activity of CAT. The level of GSH was only altered by 2,4-dichlorophenacyl derivatives. It was observed that increasing number of bromine atoms in the side chain of those derivatives was associated with decreased GSH level.

The effect of bromfenvinphos, its impurities and chlorfenvinphos on acetylcholinesterase activity.

The aim of this work was to examine the effect of two organophosphorous compounds i.e. bromfenvinphos (BFVF) and chlorfenvinphos (CFVF) possessing acaricidal and insecticidal properties, on the activity of human erythrocytes acetylcholinesterase (AChE, EC 3.1.1.7). Moreover, the effect of five bromfenvinphos production impurities on AChE activity was studied. The erythrocytes were incubated with the compounds studied in the concentrations range from 0.05 to 250 µM for 1h. The organophosphorous compounds studied in low concentrations increased Km value but they did not change Vmax value (competitive inhibition). Higher concentrations of the compounds studied decreased Vmax value and increased Km value, what revealed a mixed type of AChE inhibition by these xenobiotics. Basic significance in AChE activity inhibition has the type of halogen in vinyl group. Chlorfenvinphos (insecticide) exhibited stronger enzyme inhibition than bromfenvinphos. CFVF and dibromo-bromfenvinphos possessed the lowest Ki and Ki' values among all the compounds studied. The presence of Cl atom (chlorfenvinphos) instead of Br atom (bromfenvinphos) considerably increases antiesterase activity of the individual compound. Three impurities like 2,4-dichlorophenacyl bromide, 2,4-dichlorophenacylidene bromide and 2,4-dichlorophenacylidyne bromide did not induce any statistically changes in AChE activity. Two impurities of bromfenvinphos such as: dihydro-bromfenvinphos and dibromo-bromfenvinphos revealed significant effect on the AChE activity, which may be connected with the presence a phosphate group in these substances. It was proven that an increase in antiesterase activitiy of the compounds studied corresponded with the increase in the number of Br atoms at carbon of their vinyl group: dibromo-bromfenvinphos>bromfenvinphos>dihydro-bromfenvinphos.

The effect of bromfenvinphos and its impurities on human erythrocyte.

Bromfenvinphos - (E,Z)-O,O-diethyl-O-[1-(2,4-dichlorophenyl)-2-bromovinyl] phosphate (BFVF) is the insecticide elaborated in Poland, which has been used against Varroa destructor causing honey bees disease called as varroosis. The substances that are formed as a result of bromfenvinphos synthesis are dihydro-bromfenvinphos (O,O-diethyl O-[1-(2,4-dichlorophenyl)vinyl] phosphate); dibromo-bromfenvinphos (O,O-diethyl O-[1-(2,4-dichlorophenyl)-2,2-dibromovinyl] phosphate); 2,4-dichlorophenacyl bromide; 2,4-dichlorophenacylidene bromide and 2,4-dichlorophenacylidyne bromide. In this work, we evaluated the effect of these compounds on hemolysis and hemoglobin oxidation (met-Hb formation) in human erythrocytes. Moreover, the changes in the size (FSC-A) and the shape (SSC-A) of red blood cells were assessed using flow cytometry and phase contrast microscopy. It was proven that bromfenvinphos at concentrations ranging from 0.5 to 250 µM during 1h incubation did not change the parameters examined in human erythrocytes. Similarly, most of bromfenvinphos impurities did not increase hemolysis and methemoglobin level nor changed the size and shape of the erythrocytes. The exception was dibromo-bromfenvinphos, which changed the FSC-A and SSC-A parameters, as well as 2,4-dichlorophenacyl bromide which induced hemolysis, increased the level of met-Hb and changed erythrocytes morphology.

Ionic liquids for the production of insecticidal and microbicidal extracts of the fungus Cantharellus cibarius.

Different ionic liquids were used as solvents for the effective extraction of the active metabolites of the fruit bodies of C. cibarius. The type of ionic liquid was found to play a significant role in this process. We found that the protic ionic liquid 1-[(nonyloxy)methyl]-1H-imidazol-3-ium salicylate (6) is a most-efficient extracting agent, being superior to classical solvents such as AcOEt or hexane. The obtained extracts generally revealed high insecticidal activities against both house fly and cockroach, with similar potencies as the standard pesticides bromfenvinphos or alphacypermethrin, as well as significant activities against bacteria, yeast, and moulds. Notably, the cidal activities against plant-pathogenic bacteria were stronger than against human bacterial strains.

Influence of bromfenvinphos alone, and in mixture with methoxychlor, on levels of gamma-glutamyl transpeptidase, ceruloplasmin and cholesterol in the blood plasma of laboratory mice.

Bromfenvinphos [0,0-diethyl-1-(2,4-dichlorophenyl)-2-bromovinyl phosphate], (BrV), 12.33 mg/kg/day alone and in combination with methoxychlor [1,1,1-trichloro-2,2-bis(4-methoxyphenyl)ethane], (MeOCl), 24.66 mg/kg/day were administered daily per os (intragastrically) in pure olive-oil for 6 weeks to male laboratory mice. The activity of gamma-glutamyl transpeptidase (GGTP), ceruloplasmin (CRP), and cholesterol in the blood plasma were determined. The hematocrit value, the erythrocyte count and the weights of the liver, spleen, heart, kidney and gonads were also measured after the end of treatment. BrV and BrV + MeOCl increased the activity of gamma-glutamyl transpeptidase. The activity of ceruloplasmin and the level of cholesterol in the blood plasma as well as the erythrocyte count in the blood were also similar in the control and experimental mice. The hematocrit value decreased after BrV and MeOCl administration. The relative weight of liver was higher in mice receiving both drugs in combination, than in control and in mice receiving BrV alone. The relative weight of spleen was significantly higher in animals receiving BrV alone, than in other groups.

Hepatic and renal ultrastructural changes in cockerels exposed to cadmium chloride and subsequent interaction with organophosphate insecticide.

Ultrastructural alterations of the liver and kidneys of cockerels exposed to 100 ppm of cadmium chloride (CdCl2) and subsequent interaction with an organophosphorus compound (methylobromofenvinphos) were studied. Four groups, each consisting of 25 birds, included 100 ppm of CdCl2 in drinking water for 4 weeks (Group A), 100 ppm of CdCl2 for 4 weeks followed by a single dose of 240 mg/kg of methylobromofenvinphos (IPO 63 compound) (Group B), single dose of 240 mg/kg of IPO 63 compound (Group C), and untreated control (Group D). Three birds from each group were sacrificed 24 hr post treatment with IPO 63 compound and tissue pieces were collected for electron microscopic study. Ultrastructural changes in hepatocytes included swollen mitochondria with cavitation, dilated rough endoplasmic reticulum, numerous lysosomal bodies, myelin figures, depletion of glycogen granules, and numerous vacuoles containing degenerated membranes in birds that interacted with CdCl2. In a few cells the nuclei were markedly damaged with dilation of envelope. Renal corpuscles of CdCl2 showed irregular foot processes and thickening of the glomerular basement membrane. The proximal tubular cells of CdCl2 birds showed marked ultrastructural alterations, including numerous lysosomal bodies, few fat droplets, membrane bound vacuoles studded with polyribosomes, swollen mitochondria with fragmented cristae surrounded by rough endoplasmic reticulum, vacuoles containing myelin figures, and damaged nuclei with dilated envelope. Minor ultrastructural alterations were observed in the liver and kidneys of birds treated with cadmium alone and of those treated only with IPO 63 compound. These observations suggest that treatment with CdCl2 and then subsequent interaction with IPO 63 compound causes hepatic and renal damage that appears to be additive.

Ultrastructural alterations produced in cockerels after mercuric chloride toxicity and subsequent interaction with an organophosphate insecticide.

Ultrastructural changes of liver and kidneys of cockerels exposed to mercuric chloride and subsequent interaction with methylobromofenvinphos (IPO 63 compound) were studied. Group A birds were treated for 4 weeks with 300 ppm mercuric chloride in drinking water; Group B birds were treated for 4 weeks with mercuric chloride followed by single oral dose of 240 mg/kg of IPO 63; Group C 240 mg/kg IPO 63 only; and Group D, unexposed controls. Hepatocytes of mercury-IPO 63 interaction group B showed large lysosomes containing myelin bodies, swollen mitochondria with cristeolysis, dilated endoplasmic reticulum and numerous vacuoles containing granular material. Mercury-intoxicated birds showed similar but less severe changes, whereas IPO 63-treated birds showed accumulation of glycogen granules, fat droplets, and few lysosomal bodies as well as other changes. Renal corpuscles of kidney from mercury-IPO 63 interaction birds revealed minor ultrastructural changes as vacuolation, swollen mitochondria of podocytes and slight thickening of the glomerular basement membrane. Proximal tubular cells showed extreme damage such as, microvillar loss, dilation of endoplasmic reticulum, accumulation of lysosomal bodies, glycogen granules, myelin figures, swollen mitochondria with granular material, numerous vacuoles containing degenerated membranous organelles and distorted, pyknotic nucleus with marked dilation of nuclear membrane. Mercury intoxicated birds showed similar but less pronounced changes in tubules. These observations suggest that the effect of mercuric chloride toxicity and then interaction with an organophosphorus insecticide causes extreme damage to hepatic and renal cells that appears to be additive.

Dynamics of excretion (14C) bromfenvinphos in rats and dogs.

The dynamics of excretion of 14C in rat and dog after a single oral administration of 14C-bromfenvinphos was investigated. The level of radioactivity in urine, faeces and expired air in rats and in urine and faeces in dogs and the level of 14C in rat, depending on the dose and on the position of the labeling of the bromfenvinphos molecule (14C-vinyl) and (14C-ethyl), were determined. Also, the decay of 14C in blood and the retention degree of radioactivity in selected rat tissues were examined.

[Effect of pesticides on selected biochemical parameters in the serum and liver of rats]. / Wplyw pestycydów na wybrane parametry biochemiczne w surowicy i watrobie u szczurów.

The experiments were carried out on male Wistar rats, weighing 200-250 g. The animals received oil or oily solutions of carbaryl, parathion-methyl and IPO62 in doses of 0.5, 0.1 or 0.02 LD50. 1, 2, 4 or 24 h after the administration of pesticides, the activities of ChE and blood glucose level, liver glycogen level, FFA and TG concentrations in serum and TG level in liver were estimated. Comparing the enzymatic changes in acute intoxications with carbaryl, parathion-methyl or IPO62, in the same doses, it was found that mechanisms of efficacy of those compounds had different trends. Changes in the activities of such parameters as ChE in serum, BGR, AspAT and AlAT in serum and liver and blood glucose level depend on the dose of pesticides and this effect is observed for IPO62, parathion-methyl and carbaryl.

[Effects of IPO-63, Carbaryl, Foschlor and Reglone on Newcastle disease virus replication in chick embryo and chick embryo cell cultures]. / Wplyw preparatów IPO-63, karbaryl, foschlor i reglone na namnazanie sie wirusa choroby Newcastle w zarodkach kurzych i hodowlach komórek zarodka kurzego.

Cell cultures and chicken embryos were treated with maximum tolerant concentrations of different compounds and infected with Newcastle disease virus simultaneously or 24 hours after the compounds were introduced. The similar results were obtained in both cases. It was found that Reglone inhibited Carbaryl increased virus multiplication. The study on dynamics of virus multiplication indicates that only in the case of IPO and Carbaryl their stimulatory effect on virus at the final stage was preceded by its inhibition.

[Effect of Polfos on the bleeding time. III. Changes in various parameters of the circulatory system]. / Wplyw Polfosu na czas krwawienia. III. Zachowanie see niektórych parametrów ukladu krazenia.

The influence of Polfos on the blood pressure in rats was measured by direct and indirect methods. The influence of this pesticide on the heart rate was also examined. Two hours after Polfos administration an increase in blood pressure and a decrease in heart rate were observed. In the late phase of Polfos-intoxication no changes in the cardiovascular system were found. The results show that prolonged bleeding time in the initial phase of the intoxication might depend on its influence on the cardiovascular system in rats.

[Effect of Polfos on the bleeding time. II. Changes in selected parameters of the blood coagulation system and fibrinolysis]. / Wplyw Polfosu na czas krwawienia. II. Zachowanie sie wybranych parametrów ukladu krzepniecia i fibrynolizy.

The influence of Polfos on some parameters of the coagulation system and fibrinolysis in rats was examined. The present results indicate that Polfos had no effect on the coagulation system and fibrinolysis in the initial phase of intoxication (2 hours). In the late phase of intoxication (24 hours) a decrease in the prothrombin index, shortening of thrombin time, increase in the fibrinogen level and prolongation of euglobulin lysis time were observed. The results demonstrate that the normalization of the bleeding time in the late phase of intoxication depended on activation of the coagulation system and inhibition of fibrinolysis activation.

[Effect of Polfos on bleeding time. I. Changes in the formed elements of the blood in rats exposed to Polfos]. / Wplyw Polfosu na czas krawawienia. I. Zachowanie sie elementów morfotycznych krwi szczurów eksponowanych na Polfos.

The influence of Polfos and its metabolite, 2,4 dichlorophenacyl bromide, on the bleeding time, count of platelets, platelets aggregation, count of erythrocytes, level of haemoglobin and count of leucocytes in rats were studied. The data indicate that Polfos prolonged the bleeding time in the 2nd hour after its administration. A decrease of the platelets count was observed only in the initial phase of the intoxication. In the study the platelets count, red cells count, hematocrit and the level of haemoglobin increased 24 hours after the administration of the pesticide. The results demonstrate that the prolongation of the bleeding time in the rats after the administration of Polfos partly depends on the changing of the platelets count.

The effect of methylbromophenvinfos (Polfos) on some enzymes in vivo and in vitro. I. In vivo studies.

The experiment was carried out on Wistar rats receiving orally either oil or oily solution of methylbromophenvinfos (Polfos) either in a single dose of 0.5 LD50, or doses of 0.1 LD50 once daily for a period of 2, 4 or 6 weeks. The activities of cholinesterase (ChE), beta-glucuronidase (beta-glu), lipase and amylase were assayed in the blood serum, the activity of acetylcholinesterase (AChE)-in brain homogenates, and the activities of lipase and amylase-in homogenates of the pancreas. Cholinesterases were inhibited in the course of both acute and chronic poisoning with Polfos. During the acute poisoning a sharp increase in the activity of beta-glu in the blood serum, 1 and 2 h after the pesticide administration, was observed. Polfos inhibited lipase and amylase both after acute and chronic treatment.

Sites of methylation of DNA bases by the action of organophosphorus insecticides in vitro.

Methylation in vitro of DNA by three methyl-14C-labelled organophosphorus insecticides has been studied. The ability of methylbromphenvinphos, methylparathion and malathion to methylate N-7 of guanine in DNA can be expressed as 100:40:15. Among the methylation products, no O6-methylguanine, a known mutagen, was found. Both in the reaction with dsDNA and with ssDNA 7-methyl-guanine was the main methylation product. However, all methyl derivatives of adenine (3-methyladenine, 1-methyladenine and 7-methyladenine) constituted about 40% and 50% of all methylation products in the case of dsDNA and ssDNA, respectively. The only methyl derivative of pyrimidine we have identified was 3-methylcytosine. In the case of dsDNA 3-methylcytosine appeared in small amounts but in the alkylated ssDNA 3-methylcytosine C constituted about 20% of all alkylation products.