Pitfalls when determining tissue distributions of organophosphorus chemicals: sodium fluoride accelerates chemical degradation.
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This paper describes the tissue distributions of dichlorvos, an organophosphate, and chlorpyrifos-methyl, an organophosphorothioate, in a male individual who died after ingesting an insecticidal preparation containing these chemicals and the results of an in vitro stability study on dichlorvos and chlorpyrifos-methyl in blood and buffers. Tiny amounts of dichlorvos, 0.067 and 0.027 mg/L, were detected in the vitreous humor and cerebrospinal fluid, respectively. Although dichlorvos (0.082-8.99 mg/L or mg/kg) was detected in the thoracic aortic blood, thoracic inferior vena caval blood, pericardial fluid, bile, and spleen, it was strongly suggested that it had diffused postmortem from the stomach, which contained 879 mg, because no dichlorvos was detected in the other blood samples and tissues tested. Substantial amounts (0.615-4.15 mg/L) of chlorpyrifos-methyl were detected in all blood samples, and the order of its concentrations was as follows: pulmonary vessel blood > thoracic inferior vena caval blood > blood in the right cardiac chambers > blood in the left cardiac chambers approximately thoracic aortic blood > right femoral venous blood. The total amount of chlorpyrifos-methyl in the stomach was 612 mg. However, it was strongly suggested that virtually no chlorpyrifos-methyl diffused from the stomach into surrounding fluids and tissues postmortem because no chlorpyrifos-methyl was detected in the bile and little was found in the pericardial fluids. Neither compound was detected in the urine. In vitro experiments showed that dichlorvos (10 mg/L) almost disappeared from fresh (pH 7.4) and acidified (pH 6.2) blood samples within 24 and 72 h, respectively. However, 53 and 77% of the original amount of dichlorvos in 0.05M phosphate buffers at pH 7.4 and 6.2 were detected 72 h later. Chlorpyrifos-methyl (1 mg/L) was very stable in blood samples, regardless of the pH, during the 72-h study period, but in the pH 7.4 and 6.2 phosphate buffers, approximately 80% of the original amount had degraded after 72 h. These results indicate that organophosphates are degraded more rapidly by esterase activities than by chemical mechanisms and that organophosphorothioates are hydrolyzed chemically in aqueous solutions but are very stable in biological specimens and not metabolized by esterases. When sodium fluoride was added to blood samples, dichlorvos degraded completely within 15 min, and chlorpyrifos-methyl became very unstable. Thus, when analyzing samples to detect organophosphorus chemicals, this common preservative should not be added to fluid specimens. (+info)
Identification of acylpeptide hydrolase as a sensitive site for reaction with organophosphorus compounds and a potential target for cognitive enhancing drugs.
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We describe here the purification and identification of a previously unrecognized target for organophosphorus compounds. The target, acylpeptide hydrolase, was isolated as a tritiated-diisopropylfluorophosphate-reactive protein from porcine brain and purified to homogeneity using a combination of ion-exchange and gel-filtration chromatography. Biochemical characterization and internal sequence analysis confirmed identity. Acylpeptide hydrolase was found to be potently inhibited by the organophosphorus compounds chlorpyrifosmethyl oxon, dichlorvos, and diisopropylfluorophosphate (20-min IC(50) values of 18.3 +/- 2.0, 118.7 +/- 9.7, and 22.5 +/- 1.2 nM, respectively). The in vitro sensitivity of acylpeptide hydrolase toward these compounds is between six and ten times greater than that of acetylcholinesterase (AChE), making it a target of pharmacological and toxicological significance. We show that, in vivo, acylpeptide hydrolase is significantly more sensitive than AChE to inhibition by dichlorvos and that the inhibition is more prolonged after a single dose of inhibitor. Furthermore, using dichlorvos as a progressive inhibitor, it was possible to show that acylpeptide hydrolase is the only enzyme in the brain capable of hydrolyzing the substrate N-acetyl-alanyl-p-nitroanilide. A concentration of 154 +/- 27 pmol of acylpeptide hydrolase/gram of fresh rat brain was also deduced by specific labeling with tritiated-diisopropylfluorophosphate. We also suggest that, by comparison of structure-activity relationships, acylpeptide hydrolase may be the target for the cognitive-enhancing effects of certain organophosphorus compounds. Acylpeptide hydrolase cleaves N(alpha)-acylated amino acids from small peptides and may be involved in regulation of neuropeptide turnover, which provides a new and plausible mechanism for its proposed cognitive enhancement effect. (+info)
Retention of cytoplasmic droplet by rat cauda epididymal spermatozoa after treatment with cytotoxic and xenobiotic agents.
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Spermatozoa leaving the testis contain a cytoplasmic droplet which they release during transit through the epididymis before reaching the cauda epididymidis. The cytoplasmic droplet shows P450 aromatase activity, which plays a role in synthesis of oestrogen from androgen. In the present study, 3-month-old Wistar strain male albino rats were administered with the organophosphate insecticides malathion or dichlorvos, or the phytotherapeutics andrographolide or ursolic acid. Segments of the epididymis were subjected to histopathological and ultrastructural analyses and it was found that 60-95% of the spermatozoa residing in the lumen of the cauda epididymidis retained the cytoplasmic droplet. The motility of the spermatozoa released from the cauda epididymidis was inhibited. One of the mechanisms of action of these toxicants on male reproductive function may be attributed to the retention of the cytoplasmic droplet and the resultant impairment of sperm motility. (+info)
Effects of S-ethyl hexahydro-1H-azepine-1-carbothioate (molinate) on di-n-butyl dichlorovinyl phosphate (DBDCVP) neuropathy.
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Certain esterase inhibitors protect from organophosphate-induced delayed polyneuropathy (OPIDP) when given before a neuropathic organophosphate by inhibiting neuropathy target esterase (NTE). In contrast, they can exaggerate OPIDP when given afterwards and this effect (promotion) is associated with inhibition of another esterase (M200). In vitro sensitivities of hen, rat, and human NTE and M200 to the active metabolites of molinate, sulfone, and sulfoxide, were similar. NTE and M200 were irreversibly inhibited (> 78%) in brain and peripheral nerve of hens and rats given molinate (100-180 mg/kg, sc). No clinical or morphological signs of neuropathy developed in these animals. Hens and rats were protected from di-n-butyl dichlorovinyl phosphate neuropathy (DBDCVP, 1 and 5 mg/kg, sc, respectively) by molinate (180 or 100 mg/kg, sc, 24 h earlier, respectively) whereas 45 mg/kg, sc molinate causing about 34% NTE inhibition offered partial protection to hens. Hens treated with DBDCVP (0.4 mg/kg, sc) developed a mild OPIDP; molinate (180 mg/kg, 24 h later) increased the severity of clinical effects and of histopathology in spinal cord and in peripheral nerves. Lower doses of molinate (45 mg/kg, sc), causing about 47% M200 inhibition, did not promote OPIDP whereas the effect of 90 mg/kg, sc (corresponding to about 50-60% inhibition) was mild and not statistically significant. OPIDP induced by DBDCVP (5 mg/kg, sc) in rats was promoted by molinate (100 mg/kg, sc). In conclusion, protection from DBDCVP neuropathy by molinate is correlated with inhibition of NTE whereas promotion of DBDCVP neuropathy is associated with > 50% M200 inhibition. (+info)
Short- and long-term effect of acetylcholinesterase inhibition on the expression and metabolism of the amyloid precursor protein.
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We have investigated the acute and chronic effect of metrifonate (MTF) and dichlorvos (DDVP), respectively the prodrug and active acetylcholinesterase inhibitor, on the secretory processing of the amyloid precursor protein (APP) in SH-SY5Y neuroblastoma cells. We demonstrate that the acute treatment of SH-SY5Y cells with both compounds results in an increased secretion of the soluble fragment of APP (sAPPalpha) into the conditioned media of cells, with a pattern correlated to the level of acetycholinesterase inhibition. The regulation of APP processing in these conditions is mediated by an indirect cholinergic effect on muscarinic receptors, as demonstrated by inhibition with atropine. We have also followed APP expression and metabolism after long-term treatment with metrifonate. Treated cells showed reduced AChE activity after 24, 48 h and also following 7 days of repeated treatment, a time point at which increased AChE expression was detectable. At all time points sAPPalpha release was unaffected suggesting that enhanced sAPPalpha release by MTF is transitory, nevertheless the sensitivity of cholinergic receptors was unchanged, as indicated by the fact that cholinergic response can be elicited similarly in untreated and treated cells. APP gene expression was unaffected by long-term AChE inhibition suggesting that increased short-term sAPPalpha release does not elicit compensatory effects. (+info)
Proposal for a method for testing resistance of clothing and gloves to penetration by pesticides.
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The paper presents the proposal for a method for testing the resistance of materials used for the production of protective clothing and gloves to penetration by concentrated chemical preparations of pesticides. It has been based to a large extent on the recommendations of European standards, with certain modifications resulting from the specific properties of non-volatile and non-water-soluble biologically-active components of pesticides. These modifications primarily involved the use of a solid sorption medium and adjustment of research apparatus to the research conditions consistent with the proposed method. The results of preliminary studies on penetration of the selected biologically-active components of pesticides (dichlorvos, 2,4-D, cypermethrin, carbofuran) through the selected protective materials (fabric coated with viton and butyl on both sides, rubberised fabric, butyl rubber) are presented. The study confirmed the usefulness of the proposed method for testing the resistance of materials protecting against the effect of pesticides. (+info)
Acetylcholinesterase engineering for detection of insecticide residues.
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To detect traces of insecticides in the environment using biosensors, we engineered Drosophila acetylcholinesterase (AChE) to increase its sensitivity and its rate of phosphorylation or carbamoylation by organophosphates or carbamates. The mutants made by site-directed mutagenesis were expressed in baculovirus. Different strategies were used to obtain these mutants: (i) substitution of amino acids at positions found mutated in AChE from insects resistant to insecticide, (ii) mutations of amino acids at positions suggested by 3-D structural analysis of the active site, (iii) Ala-scan analysis of amino acids lining the active site gorge, (iv) mutagenesis at positions detected as important for sensitivity in the Ala-scan analysis and (v) combination of mutations which independently enhance sensitivity. The results highlighted the difficulty of predicting the effect of mutations; this may be due to the structure of the site, a deep gorge with the active serine at the bottom and to allosteric effects between the top and the bottom of the gorge. Nevertheless, the use of these different strategies allowed us to obtain sensitive enzymes. The greatest improvement was for the sensitivity to dichlorvos for which a mutant was 300-fold more sensitive than the Drosophila wild-type enzyme and 288 000-fold more sensitive than the electric eel enzyme, the enzyme commonly used to detect organophosphate and carbamate. (+info)
Organophosphate-induced delayed neuropathy: case report.
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Organophosphate induced delayed neuropathy (OPIDN) is an uncommon clinical condition. It occurs in association with the ingestion of great amounts of organophosphate after the stimulation of cholinergic receptor. The clinical picture is characterized by a distal paresis in lower limbs associated with sensitive symptoms. Electrodiagnostic studies show a motor axonal neuropathy. Involvement of the central nervous system may occur. We describe a 39 years-old female patient who developed hyperesthesia associated with lower limbs paresis, fourteen days after she had ingested a Dichlorvos-based insecticide. Electrophysiological study was characterized by an axonal polyneuropathy pattern. Pyramidal tract dysfunction was observed later in upper limbs. Considering that both peripheral and central nervous systems are involved we believe that the more appropriated term would be organophosphate induced delayed neuropathy (OPIDN) instead of organophosphate induced delayed polyneuropathy (OPIDP). (+info)