The effects of phenobarbital pretreatment on the metabolism and toxicity of paraoxon in the mouse.

The effects of induction of various forms of cytochromes P450 by chemicals like phenobarbital on the hepatic oxidative desulfuration and acute toxicity of the phosphorothioate insecticide parathion have been well-characterized. However, the effects of these chemicals on the metabolism and acute toxicity of the active metabolite paraoxon are less understood. In the present study, daily pretreatment of mice with phenobarbital (intraperitoneally 75 mg/kg) for up to eight days resulted in a transient increase in hepatic microsomal A-esterase activity, with a corresponding transient decrease in serum A-esterase activity (A-esterase was defined as hydrolysis of paraoxon which could be inhibited by EDTA). These alterations could be accounted for by a temporary decrease in the rate of secretion of A-esterase from liver. However, the same pretreatment resulted in a sustained protective effect against the acute toxicity of paraoxon. These data suggest that alterations in A-esterase activity as a result of phenobarbital pretreatment cannot account for the observed antagonism of the acute toxicity of paraoxon. Furthermore, these data demonstrate that the protective effect of phenobarbital pretreatment on phosphorothioate insecticides like parathion cannot be attributed exclusively to alterations in oxidative desulfuration of these compounds.

The role of calcium in the hydrolysis of the organophosphate paraoxon by human serum A-esterase.

Human serum A-esterase is a calcium-dependent enzyme that hydrolyzes the organophosphate paraoxon by an Ordered Uni Bi kinetic mechanism. Incubation of various concentrations of calcium chloride with human serum A-esterase resulted in corresponding changes in appk3 and appE for the reaction, while appk2 was unaffected. Carboxyglutamic acid (CAG) prevented calcium chloride from altering appk3, but not appE. Similarly CAG reduced the calcium-stimulated nonenzymatic hydrolysis of paraoxon, as well as the calcium-stimulated de-phosphorylation of chymotrypsin phosphorylated by paraoxon. These results suggest that calcium plays two roles in the hydrolysis of paraoxon by A-esterase. Firstly, calcium is required in order to maintain an active site. In this capacity calcium might participate directly in the catalytic reaction, or it might be required in order to maintain the appropriate confirmation of the active site. And secondly, free calcium (or calcium weakly associated with A-esterase) facilitates the removal of diethyl phosphate from A-esterase, probably by polarizing the P = O bond of the diethyl phosphate-A-esterase intermediate, thereby rendering phosphorus more susceptible to nucleophilic attack by hydroxide ions.

Kinetic mechanism of the detoxification of the organophosphate paraoxon by human serum A-esterase.

The mammalian detoxification of certain organophosphates, such as paraoxon [O,O-diethyl (p-nitrophenyl) phosphate], is catalyzed by the enzyme A-esterase. In this study, incubations of human serum in 50 mM glycine buffer (pH 10.5) with paraoxon resulted in the nonlinear production of p-nitrophenol, characterized by a rapid initial phase for the first several minutes of the incubation, followed by a second, slower phase in which the velocity approached constancy. Production of p-nitrophenol could be accurately fitted to the velocity equation for an Ordered Uni Bi kinetic mechanism with initial-burst activity, yielding estimates of appk2, appk3, and appE, for 10 human subjects. Increasing calcium concentration in the incubation resulted in increases in appk3 and appE, without affecting appk2. Conversely, addition of 1 M sodium chloride decreased the appk3 and appE, but did not alter appk2. And finally, a continuous system computer model was constructed based on the differential equations descriptive of an Ordered Uni Bi kinetic mechanism. This model accurately simulated production of p-nitrophenol from human serum, providing further support that A-esterase hydrolyzes paraoxon by an Ordered Uni Bi kinetic mechanism with initial-burst activity.