Lester G. Sultatos

Mammalian toxicology of organophosphorus pesticides

Organophosphorus compounds have been utilized as pesticides for almost five decades. They continue to be used as insecticides, helminthicides, ascaricides, nematocides, and to a lesser degree as fungicides and herbicides. While they have been and continue to be extremely useful in agricultural pest control throughout the world, their extensive use has led to numerous poisonings of nontarget species, including many human fatalities. The primary acute mammalian toxicity associated with exposure to organophosphorus pesticides results from inhibition of the enzyme acetylcholinesterase. However, other toxicities, some of which are life-threatening but not related to acetylcholinesterase inhibition, have been observed following exposure to certain organophosphorus compounds. The focus of the current review is to summarize the known effects, both cholinergic and noncholinergic, of organophosphorus pesticides in mammals. Included in this summary is a discussion of the metabolic activation of organophosphorus pesticides, since this process plays a critical role in mediating the acute toxicities of many of these pesticides.

Metabolic activation of the organophosphorus insecticides chlorpyrifos and fenitrothion by perfused rat liver

The present study was undertaken to characterize the metabolic activation of the organophosphorus insecticides chlorpyrifos [O,O-diethyl O-(3,5,6-trichloro-2-pyridyl) phosphorothionate] and fenitrothion [O,O-dimethyl O-(3-methyl-p-nitrophenyl) phosphorothionate] by intact rat liver. Single-pass perfusions of rat livers with chlorpyrifos or fenitrothion to steady state conditions resulted in the appearance of their corresponding oxygen analogs in effluent. In addition, detoxification of chlorpyrifos oxon [O,O-diethyl O-(3,5,6-trichloro-2-pyridyl) phosphate] or fenitrooxon [O,O-dimethyl O-(3-methyl-p-nitrophenyl) phosphate] by rat blood did not proceed at a rate rapid enough to prevent passage of at least some of these chemicals from liver to extrahepatic tissues, suggesting that hepatic biotransformation of chlorpyrifos and fenitrothion by rat liver results in their net activation. Although male rat livers produced more chlorpyrifos oxon and fenitrooxon from chlorpyrifos and fenitrothion, respectively, than did livers from female rats, the acute toxicities of chlorpyrifos and fenitrothion were greater in females than in males. Therefore, differences in hepatic activation of chlorpyrifos and fenitrothion in males and females cannot account for the sex differences in their acute toxicities in the rat. Finally, S-methyl glutathione and S-p-nitrophenyl glutathione were not detected in effluent or bile of livers perfused with fenitrothion, suggesting that glutathione-mediated biotransformation of this insecticide does not occur to any significant degree in intact liver.

An evaluation of the inhibition of human butyrylcholinesterase and acetylcholinesterase by the organophosphate chlorpyrifos oxon.

Acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase (EC 3.1.1.8) are enzymes that belong to the superfamily of alpha/beta-hydrolase fold proteins. While they share many characteristics, they also possess many important differences. For example, whereas they have about 54% amino acid sequence identity, the active site gorge of acetylcholinesterase is considerably smaller than that of butyrylcholinesterase. Moreover, both have been shown to display simple and complex kinetic mechanisms, depending on the particular substrate examined, the substrate concentration, and incubation conditions. In the current study, incubation of butyrylthiocholine in a concentration range of 0.005-3.0 mM, with 317 pM human butyrylcholinesterase in vitro, resulted in rates of production of thiocholine that were accurately described by simple Michaelis-Menten kinetics, with a K(m) of 0.10 mM. Similarly, the inhibition of butyrylcholinesterase in vitro by the organophosphate chlorpyrifos oxon was described by simple Michaelis-Menten kinetics, with a k(i) of 3048 nM(-1) h(-1), and a K(D) of 2.02 nM. In contrast to inhibition of butyrylcholinesterase, inhibition of human acetylcholinesterase by chlorpyrifos oxon in vitro followed concentration-dependent inhibition kinetics, with the k(i) increasing as the inhibitor concentration decreased. Chlorpyrifos oxon concentrations of 10 and 0.3 nM gave k(i)s of 1.2 and 19.3 nM(-1) h(-1), respectively. Although the mechanism of concentration-dependent inhibition kinetics is not known, the much smaller, more restrictive active site gorge of acetylcholinesterase almost certainly plays a role. Similarly, the much larger active site gorge of butyrylcholinesterase likely contributes to its much greater reactivity towards chlorpyrifos oxon, compared to acetylcholinesterase.

The role of glutathione in the detoxification of the insecticides methyl parathion and azinphos-methyl in the mouse.

The dimethyl-substituted organothiophosphate insecticides methyl parathion and azinphos-methyl are thought to undergo glutathione-mediated detoxification in mammals. In the present study, depletion of hepatic glutathione in the mouse by pretreatment with diethyl maleate potentiated the acute toxicities of methyl parathion and azinphos-methyl, whereas depletion of hepatic glutathione by pretreatment with buthionine sulfoximine did not. Furthermore incubation of 50 microM methyl parathion with mouse hepatic microsomes for 5 min in the presence of 1 mM diethyl maleate led to significantly greater (p less than 0.05) production of methyl paraoxon, compared to incubations in the absence of diethyl maleate. Conversely, 1 mM diethyl maleate had no effect on metabolic activation of azinphos-methyl by mouse hepatic microsomes, while 10 mM inhibited slightly production of azinphos-methyl oxon from azinphos-methyl. These results suggest normal levels of hepatic glutathione are not required for detoxification of methyl parathion or azinphos-methyl in the mouse. Moreover the potentiation of the acute toxicity of methyl parathion following diethyl maleate pretreatment could result, at least in part, from enhanced production of methyl paraoxon. However, diethyl maleate likely acts through another mechanism(s) as well since it did not enhance the metabolic activation of azinphos-methyl in vitro. These data raise serious doubts about the participation of glutathione in the detoxification of methyl parathion and azinphos-methyl in vivo in the mouse.

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.

Factors affecting the hepatic biotransformation of the phosphorothioate pesticide chlorpyrifos

Following single-pass perfusion of mouse livers in situ with the organophosphate pesticide chlorpyrifos, the cholinesterase inhibitor chlorpyrifos oxon could not be detected in effluent perfusate. Steady-state, with respect to chlorpyrifos, was achieved in 36-48 min, at which time the extraction ratio was 0.46. The hepatic disposition of chlorpyrifos was not affected by changes in perfusate flow rates (provided the flow rates maintained viable livers), but was altered by changes in the free fraction of chlorpyrifos within perfusate. However, under no conditions did chlorpyrifos oxon appear in effluent perfusate. Pretreatment of mice with phenobarbital enhanced the production of chlorpyrifos oxon from chlorpyrifos by mouse hepatic microsomes in vitro, but antagonized the acute toxicity of chlorpyrifos. Phenobarbital pretreatment increased the steady-state extraction ratio of chlorpyrifos to 0.94, but did not lead to the presence of chlorpyrifos oxon in effluent perfusate. Thus the enhanced hepatic detoxification of chlorpyrifos following phenobarbital pretreatment probably accounts for the antagonism of the acute toxicity afforded by phenobarbital pretreatment.

Biotransformation of the insecticide parathion by mouse brain

The acute toxicity of organothiophosphate insecticides like parathion results from their metabolic activation by cytochromes P450. The present study is directed towards the characterization of cytochrome-P450-dependent metabolism of parathion by various mouse brain regions. Intraperitoneal administration of [35S]parathion to mice led to covalently bound [35S]sulfur in various tissues, indicating their capacity to oxidatively desulfurate this insecticide. Liver contained the greatest amount of covalently bound sulfur, and brain the least. Among individual brain regions the olfactory bulb and hypothalamus possessed the highest levels of sulfur binding when expressed on a per milligram tissue basis. However, when expressed on a per brain region basis, sulfur binding was greatest within the cortex as a result of the large mass of this region, compared to the hypothalamus and olfactory bulb. Incubation of the 78,000 x g fraction of mouse brain with parathion resulted in formation of p-nitrophenol, although paraoxon could not be detected. However, given the current understanding of parathion metabolism by cytochromes P450, and given that paraoxon can rapidly disappear through phosphorylation of serine hydroxyl groups, it is reasonable to assume that at least some paraoxon was formed. Production of p-nitrophenol required NADPH and was inhibited by carbon monoxide. In vitro incubations of parathion with the 78,000 x g fraction of mouse brain indicated that the hypothalamus and olfactory bulb had the greatest capacity to produce p-nitrophenol. These results demonstrate that various mouse brain regions possess different capacities to metabolize parathion.

A Physiologically Based Pharmacokinetic Model of Parathion Based on Chemical-Specific Parameters Determined In Vitro

Physiologically based pharmacokinetic (PBPK) modeling is an approach that has been used to predict successfully the pharmacokinetic disposition of numerous foreign chemicals. The accuracy of a PBPK model is dependent on the reliability of estimates of tissue/blood distribution coefficients (Kp) and appropriate kinetic constants descriptive of the elimination of the chemical under consideration. The present study evaluates the validity of the use of Kp values and kinetic constants determined in vitro for use in a PBPK model for the organothiophosphorus insecticide parathion. Kps were determined by equilibrium dialysis, whereas Vmaxs and Kms descriptive of the biotransformation of parathion were calculated from values determined previously in this laboratory. Using these kinetic constants to calculate a hepatic extraction ratio of parathion in the mouse yielded a value identical to that observed with mouse liver perfusions in situ performed previously in this laboratory. Use of Kp values and kinetic constants determined in vitro in a PBPK model accurately simulated levels of parathion in brain, lungs, blood, and liver up to 3 h after intravenous administration of this insecticide. This study demonstrates that chemical-specific parameters determined entirely in vitro can be used to construct accurate PBPK models.

The effects of the phosphorothioate insecticide fenitrothion on mammalian cytochrome P450-dependent metabolism of estradiol.

Phosphorothioate insecticides, such as fenitrothion, are suicide substrates of cytochromes P450 (P450). These compounds undergo oxidative desulfuration by P450 resulting in the release and subsequent binding of atomic sulfur to the enzyme. Consequently, the P450-dependent metabolism of certain endogenous substrates could be inhibited by exposure to these insecticides. Formation of 2-hydroxyestradiol (2-OHE2), 4-hydroxyestradiol (4-OHE2), 16 alpha-hydroxyestrone (16 alpha-OHE1), and estriol in mammals occurs by P450-dependent hydroxylation of estradiol at various positions on the steroid nucleus. In the present study, pretreatment of male Swiss Webster mice with increasing doses of fenitrothion resulted in dose-dependent biphasic decreases in 2-OHE2 and 4-OHE2 production in mouse hepatic microsomes compared to control, with substantial decreases even at a dosage as low as 7 mg/kg. Fenitrothion pretreatment also resulted in dose-dependent biphasic increases in 16 alpha-OHE1 and estriol production, along with substantial increases in estrone formation, probably as a result of shunting from the inhibition of 2- and 4-hydroxylation. These data suggest that exposure to fenitrothion might alter estradiol metabolism by inhibition of certain P450 isozymes.

Evaluation of estimations in vitro of tissue/blood distribution coefficients for organothiophosphate insecticides.

Physiologically based pharmacokinetic (PBPK) modeling of foreign chemicals is dependent on the accurate determination of their tissue/blood distribution coefficients, Kp (partition coefficients). The present study was undertaken to evaluate the validity of the in vitro estimation of the Kp values of the organothiophosphate insecticides parathion and methyl parathion by equilibrium dialysis. Data derived from previously published studies that utilized single-pass perfusions of mouse livers in situ with parathion or methyl parathion were analyzed to determine liver/perfusate Kp values from the equation Kp = (t 1/2ss) (Q)/(0.693) (VH), where Kp is the liver/perfusate distribution ratio, t 1/2ss is the half-life for approach to steady state of the chemical, VH is the liver volume, and Q is the perfusate flow rate. Kp values for methyl parathion were calculated to be 16.4 +/- 7.5 and 9.5 +/- 2.7 (mean +/- SD) for perfused livers and equilibrium dialysis, respectively, while estimates of Kp for parathion were 15.6 +/- 6.3 and 19.5 +/- 5.5 for perfused livers and equilibrium dialysis, respectively. These results indicate that equilibrium dialysis can be utilized to give an accurate estimate of tissue partitioning of parathion and methyl parathion from perfusate into perfused mouse livers.