T.R. Fukuto

Mode of action of the delayed toxicity of O,O,S-trimethyl phosphorothioate in the rat☆

Abstract As preliminary probes to determine the mode of delayed toxic action of O,O,S-trimethyl phosphorothioate (I) in the rat, the effect of I on rat tissue and organs, and on blood, urine, and pharmacokinetic parameters was investigated. Following oral administration, 30 to 200 mg/kg I caused liver necrosis as a major pathological effect. Morphological changes were also observed in the heart, adrenal, tissues of the small intestine, and kidney. Most animals treated with I developed bronchopneumonia after 3 days. Blood samples taken from rats poisoned with 60 mg/kg I showed severe hemoconcentration; however, serum Na+, Cl−, albumin, and total carbonate/bicarbonate varied only slightly. Na+ and Cl− concentrations in the urine showed a steady decline with time following poisoning but K+ levels remained relatively constant. Pharmacokinetic studies showed that 14C levels in the blood following intraperitoneal or intravenous administration of 60 mg/kg [CH3O14C]I were not affected when the animals were cotreated with 5% of the antagonist O,O,O-trimethyl phosphorothioate. However, lower levels of 14C were found in antagonized animals following oral administration.

Metabolism of O,S-dimethyl propionyl- and hexanoylphosphoramidothioate in the house fly and white mouse

Abstract The metabolism of O,S -dimethyl propionyl- and hexanoylphosphoramidothioate was investigated in the white mouse and house flies. Compared to the hexanoylphosphoramidothioate, the propionyl analog is approximately 35-fold more toxic to house flies and is 10-fold less toxic to mice. On a percentage basis, substantially larger amounts of methamidophos were detected in house flies treated topically with the propionylphosphoramidothioate than in flies treated with the hexanoyl derivative. The reverse was evident in the case of the mouse where much larger amounts of methamidophos were formed after oral treatment with the hexanoylphosphoramidothioate. Minor amounts of other metabolic products also were detected, including an unknown from the hexanoylphosphoramidothioate. Metabolism of the S -methyl moiety to carbon dioxide appeared to be a major pathway for metabolic degradation of both compounds in both the white mouse and house fly. The difference in toxicity of the two acylphosphoramidothioates to the mouse and house fly is attributed to difference in the amounts of methamidophos formed in the animals.

Insecticidal properties, antiesterase activities, and metabolism of methamidophos

Abstract Methamidophos is highly toxic to insects but at best is a moderate cholinesterase inhibitor. Evaluation of the kinetics of its housefly cholinesterase inhibition showed that its affinity for the enzyme and its phosphorylation and bimolecular inhibition rates are all relatively low. In vivo metabolism studies in houseflies provided evidence that it is not activated to a more effective cholinesterase inhibitor and indirect evidence also was obtained for its slow degradation. In vitro metabolism studies in housefly and mouse tissues provided additional evidence for its lack of activation and slow metabolic degradation. Compared to other effective organophosphorus insecticides, methamidophos was slow in producing acute symptoms of poisoning and cholinesterase inhibition and required the accumulation of comparatively high internal levels for toxic effects. However, in vivo cholinesterase inhibition studies provided evidence for the interrelationships of cholinesterase inhibition and toxic effects. Thus, its relative stability and low in vivo degradation appeared to be of critical importance in accumulating and maintaining a sufficient internal concentration for a sufficiently long period of time to permit the development of its slowly expressed toxicity.

Affinity and phosphonylation constants for the inhibition of cholinesterases by the optical isomers of O-2-butyl S-2-(dimethylammonium)ethyl ethylphosphonothioate hydrogen oxalate

Abstract The synthesis of the four optical isomers of known absolute configuration of O -2-butyl S -2-(dimethylammonium)ethyl ethylphosphonothioate hydrogen oxalate is described. Values for the affinity constant ( K a ), phosphonylation constant ( k p ), and bimolecular inhibition rate constant ( k i ) for the inhibition of bovine erythrocyte acetylcholinesterase, housefly-head acetylcholinesterase, and horse serum cholinesterase by the chiral isomers and the racemic mixture are reported. Using a relatively simple spectrophotometric technique, inhibition times as low as 0.5 sec were used. The phosphorus isomers of S p configuration were more potent inhibitors than their R p enantiomers by 1630-fold against the bovine enzyme, 9120-fold against the fly-head enzyme, and 40-fold against the horse serum enzyme. The differences in anticholinesterase activity were attributable to differences in the affinity constant, K a , and the phosphonylation constant, k p . Small but consistent inhibition rate differences were attributable to asymmetry at carbon. Against horse serum cholinesterase, the S C isomers indicated the presence of three kinetic forms in this enzyme preparation.

Biochemical and physiological investigations into the delayed toxicity produced by O,O,S-trimethyl phosphorothioate in rats

Abstract Oral administration of O,O,S -trimethyl phosphorothioate (OOS), an impurity in several technical organophosphorus insecticides, causes delayed toxicity in rats with death occurring up to 28 days after the treatment. The oral LD 50 was determined to be 60 mg/kg. The effect of a single nonlethal dose of OOS (20 mg/kg) on in vivo protein synthesis in different organs was determined by measurement of the incorporation of [ 14 C]leucine at 6 hr to 28 days after treatment. As early as 6 hr after OOS treatment the incorporation of [ 14 C]leucine into the liver, lung, thymus, kidney, and spleen was elevated and remained elevated for up to 7 days. With the exception of the lung, organ weights were significantly decreased during the same time period. On Day 28 after treatment, the amount of [ 14 C]leucine incorporation had decreased to the control level in all of the organs studied. Treatment with OOS at 20 mg/kg caused a significant increase in hematocrit on Days 3,5, and 7, and as early as 6 hr after treatment at 60 mg/kg. The clinical biochemistry of plasma indicated that there was no significant change from control values in the glutamic pyruvic transaminase, glutamic oxalic transaminase, lactate dehydrogenase, or alkaline phosphatase activities with the 20 mg/kg dose. The analysis of the intermediary metabolites indicated that the redox state of cytosol was more reduced on Day 5, whereas that of mitochondria was not affected by OOS. Data obtained at selected times after oral administration of a 60 mg/kg dose of OOS and that obtained from animals starved for 3 days are also discussed.

Metabolism of 2,2-dimethyl-2,3-dihydrobenzofuranyl-7 N-methyl-N-(2-toluenesulfenyl)carbamate in the housefly and white mouse

Abstract The metabolism of a new, selectively toxic derivative of carbofuran, 2,2-dimethyl-2,3-dihydrobenzofuranyl-7 N-methyl-N-(2-toluenesulfenyl)carbamate has been investigated. The selective toxicity between insect and mammal is due to differing pathways of metabolism. Houseflies appear peculiarly suited for the rapid liberation of the toxic agent, carbofuran, from N-(2-toluenesulfenyl) carbofuran in large amounts. Metabolism in the mouse is more complex and involves a series of oxidative and hydrolytic detoxication processes which do not result in the formation of carbofuran.

Delayed acute toxicity of O,S,S-trimethyl phosphorodithioate and O,O,S-trimethyl phosphorothioate to the rat

Abstract The toxicity and LD 50 of O,S,S -trimethyl phosphorodithioate were reexamined in the rat. Animals treated orally (single dose) with this compound exhibited early cholinergic signs followed at approximately 5 hr by delayed toxic signs, with an LD 50 of 43 mg/kg. Contamination of O,S,S -trimethyl phosphorodithioate by as much as 5% (w/w) O,O,O -trimethyl phosphorothioate provided only limited antagonism to the dithioates toxicity. In contrast, the addition of 5% O,O,S -trimethyl phosphorodithioate to O,O,S -trimethyl phosphorothioate gave protection against the toxic effects of the latter compound up to 80 mg/kg of toxicant. Pretreatment of rats with as little as 5% O,O,O -trimethyl phosphorothioate, 24 hr prior to treatment with 200 mg/kg O,O,S -trimethyl phosphorothioate, gave complete protection against the toxic effects of this compound. Conversely, administration of 10% (w/w) O,O,O -trimethyl phosphorothioate 4 or 24 hr after treatment with 60 or 80 mg/kg of O,O,S -trimethyl phosphorothioate provided only partial protection at 4 hr and no protection from the effects of the toxicant at 24 hr. The ability of O,O,O -trimethyl phosphorothioate to antagonize the toxicity of this compound depended markedly on the route of administration (oral, intravenous, or intraperitoneal). At 4 hr past treatment with toxicant, only oral administration of the antagonist provided full protection. Intraperitoneal and intravenous administration of antagonist 4 hr after treatment with toxicant were partially effective and completely ineffective, respectively, in halting the toxic effects of this compound.

Metabolism of O,O,S-trimethyl phosphorothioate in rats after oral administration of a toxic dose, and the effect of coadministration of its antagonistic thionate isomer

Abstract The in vivo metabolism of [14CH3S]- and [14CH3O]O,O,S-trimethyl phosphorothioate (OOS) was followed in rats after oral administration of threshold or LD50 toxic doses of 20 or 60 mg/kg. Similar metabolic studies were conducted with coadministration of 1% O,O,O-trimethyl phosphorothionate (OOO), which prevented all signs of delayed toxicity, including weight loss. When administered alone, OOS was metabolized mainly (50–60%) via removal of the CH3S moiety, which was largely converted to expired CO2. Approximately 20% of the compound was O-demethylated, presumably by conjugation with glutathione, and then further metabolized to CO2. Major urinary products were identified as O,O-dimethyl phosphoric acid (50–60%) and O,S-dimethyl phosphorothioic acid (∼20%). Coadministration of OOO caused a slight decrease (∼5%) in the cleavage of the CH3S moiety, indicated by a reduction in 14CO2 from [14CH3S]OOS and a quantitatively similar increase in the formation of O,S-dimethyl phosphoric acid. Limited pharmacokinetic studies indicated that OOS was rapidly absorbed and distributed throughout the body. Coadministration of 1% OOO caused a slight increase in the blood half-life of parent OOS when administered at 60 mg/kg. It was concluded that a small proportion of the cleavage of the CH3S moiety from OOS is involved in the intoxication process, and that this intoxication reaction is specifically inhibited by OOO.

Thiolysis as an activation process in N-sulfenylated derivatives of methylcarbamate esters

Abstract An activation process involving thiolytic cleavage of the NS bond in several N -sulfenylated- N -methylcarbamates was shown to be a nonenzymatic reaction. Biological tissues, including subcellular fractions of mouse liver; heat-denatured microsomal enzyme, mouse blood, commercial protein, and housefly homogenate, as well as thiol reagents, readily effected the cleavage of the NS bond. The inhibitory effect of sulfhydryl inhibitors such as N -methylmaleimide and p -chloromercuribenzoate in the incubation system suggests that thiol residues in biological tissue are involved in this thiolytic reaction. Results from kinetic and product analysis also support this conclusion.

The reactivation of carbamate-inhibited cholinesterase, kinetic parameters

Abstract The rates of spontaneous regeneration or decarbamylation of fly-head and bovine erythrocyte cholinesterase inhibited by methyl- and dimethylcarbamic acid esters were determined under different conditions of pH, salt concentrations, and temperature using Sephadex gel filtration as a means of isolating the carbamylated enzyme. The pH-rate profiles for decarbamylation of the two cholinesterases for both methyl- and dimethylcarbamylated enzyme had maximum rates at pH 8.5–8.9 for bovine erythrocyte and pH 8.1–8.5 for fly-head cholinesterase. Imidazole, hydroxylamine, and inorganic salts did not alter the decarbamylation rate. Values for the energy of activation for the decarbamylation reaction indicate a somewhat unstable and more reactive carbamylated enzyme for bovine cholinesterase compared to fly-head cholinesterase, but entropy factors are more favorable for decarbamylation in the latter. Regeneration rates in deuterium oxide for both enzymes and both methyl- and dimethylcarbamates were approximately six to seven times slower than in water, indicating a secondary isotope effect. A mechanism for decarbamylation consistent with the data is cited.