Comparison of Esterase Sensitivity, Metabolic Efficiency, and Toxicity

Chapter 12

Downloaded by COLUMBIA UNIV on July 26, 2012 | http://pubs.acs.org Publication Date (Web): July 25, 2012 | doi: 10.1021/bk-2012-1099.ch012

Comparison of Esterase Sensitivity, Metabolic Efficiency, and Toxicity Levels of Two Organophosphorus Insecticides: Parathion and Chlorpyrifos Janice E. Chambers,*,1 Edward C. Meek,1 and Howard W. Chambers2 1Center

for Environmental Health Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, Mississippi 39762 2Center for Environmental Health Sciences, Department of Biochemistry, Molecular Biology, Entomology and Plant Pathology, Mississippi State University, Mississippi State, Mississippi 39762 *E-mail: [email protected]

Parathion and chlorpyrifos are phosphorothionate insecticides, and parathion is about an order of magnitude more toxic than chlorpyrifos. Our laboratories have investigated several aspects of the biochemical reactivity and the metabolism of these two insecticides in rats to identify factors that influence the acute toxicity level. Both insecticides are bioactivated by cytochromes P450 to potent oxon metabolites by desulfuration, with bioactivation of parathion more efficient than that of chlorpyrifos. P450-mediated detoxication, dearylation, can also occur, and is more effective against chlorpyrifos than parathion. The oxons can persistently inhibit serine esterases, including nervous system acetylcholinesterase (the target enzyme for acute toxicity) and protective B-esterases such as carboxylesterases; chlorpyrifos-oxon is a more potent inhibitor of both acetylcholinesterase and carboxylesterases © 2012 American Chemical Society In Parameters for Pesticide QSAR and PBPK/PD Models for Human Risk Assessment; Knaak, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


than paraoxon. The oxons can be hydrolyzed by paraoxonase (PON) which is much more efficient towards chlorpyrifos-oxon than paraoxon. Chlorpyrifos, because of its high lipophilicity, results in more persistent inhibition of serine esterases than does parathion. The lower toxicity of chlorpyrifos than parathion seems to be the result largely of less effective bioactivation and more effective detoxication by P450, carboxylesterases and paraoxonase, despite the greater affinity of chlorpyrifos-oxon than paraoxon toward acetylcholinesterase. These factors can be useful in PBPK modeling.

Introduction The organophosphorus (OP) insecticides are among the most widely used of the synthetic organic insecticides, and have been in use for 50-60 years. As a class they display a wide range of toxicity levels, with some like parathion and methyl parathion displaying high acute toxicity levels (rat oral LD50’s, 2-13 and 6 mg/kg, respectively), some like chlorpyrifos and diazinon displaying intermediate acute toxicity levels (rat oral LD50’s, 82-155 and 1250 mg/kg, respectively), while others like malathion displaying very low acute toxicity levels (rat oral LD50, 5500 mg/kg) (1–3). The mechanism of acute toxicity is well established to be the persistent inhibition of acetylcholinesterase (AChE) which leads to an accumulation of the neurotransmitter acetylcholine in synapses and neuromuscular junctions, resulting in mammals in hyperexcitability within the nervous system leading to tremors, convulsions, the SLUD syndrome (salivation, lacrimation, urination and defecation), bronchiolar constriction, paralysis of the respiratory muscles and eventually respiratory failure in cases of lethal dose poisonings (4). While other mechanisms of toxicity may be at play in other types of organophosphorus compound toxicity, the perspective of this paper is the acute toxicity brought on by AChE inhibition, and the factors influencing the acute toxicity level. Our laboratories have many years’ experience studying these various factors, and have characterized several of these factors as important influences on the acute toxicity levels. Our emphasis has been on comparing two important organophosphorus insecticides: parathion (O,O-diethyl O-4-nitrophenyl phosphorothioate), a high toxicity insecticide developed early during the history of OP insecticides which, in many regards, has been viewed as the prototypical OP insecticide, and whose use is highly restricted now because of its high toxicity; and chlorpyrifos (O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphorothioate), a moderate toxicity insecticide which has been very widely used both residentially and agriculturally, although most residential uses of chlorpyrifos have been eliminated in recent years. An overview of OP compound chemistry, metabolism and toxicity can be found in Chambers and Levi (5). 180 In Parameters for Pesticide QSAR and PBPK/PD Models for Human Risk Assessment; Knaak, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Many of the OP insecticides, including the two of interest here, are phosphorothionates, which are characterized by the presence of a sulfur on the pentavalent central phosphorus atom which is attached by a coordinate covalent bond, typically drawn as a double bond. The other three valences are groups, usually organic, bonded by single bonds, with one of the three groups considered the “leaving group”, and it is the moiety most likely to be removed (“leave”) when the compound phosphorylates an enzyme. The phosphorothionates are relatively weak anticholinesterases and require metabolic activation to their oxon metabolites, which are potent anticholinesterases, in order to display appreciable acute toxicity (and probably many of the other OP compound toxicities as well). In the case of parathion, the difference in potency between the parent phosphorothionate, parathion, and its oxon metabolite, paraoxon, in anticholinesterase potency is three orders of magnitude, indicating the importance of the bioactivation reaction (6). In most cases, and certainly with parathion and chlorpyrifos, the bioactivation is mediated by cytochromes P450 (CYPs) (7, 8). However, CYPs can also mediate a detoxication reaction on the phosphorothionates, a reaction termed dearylation because the leaving group, which is frequently aromatic, is removed; while this appears on the surface to be a hydrolysis, it is a CYP-mediated reaction requiring molecular oxygen and NADPH as a source of reducing equivalents. The oxons can be detoxified by both catalytic and stoichiometric mechanisms (9, 10). Catalytically the A-esterases (also named paraoxonase) are calcium-dependent hydrolases which can hydrolyze oxons into the leaving group and, in the case of our two example compounds, diethyl phosphate. The oxons are potent inhibitors of serine esterases, and can stoichiometrically phosphorylate serine esterases, including butyrylcholinesterase, carboxylesterases and non-target erythrocyte AChE. Phosphorylation of any of these non-target esterases destroys the oxon, but since the phosphorylation is persistent, there is little turnover, so this mechanism is not considered to be catalytic. Any oxon escaping the above detoxication mechanisms is available to inhibit target AChE in synapses and neuromuscular junctions and to exert acute toxicity. These metabolic and toxicity relationships are illustrated in Figure 1. All or a portion of these pathways have been described numerous times, and a few of the earlier references are cited (6–10). The perspective of this paper is to describe the efficiency or the sensitivity of these several processes to better understand the biochemical factors influencing the acute toxicity levels of these two phosphorothionate insecticides. These biochemical factors, once measured as various rate constants or inhibition constants, are then useful factors to be placed into physiologically-based pharmacokinetic (toxicokinetic) models (11, 12). Our emphasis in this article will be a comparison of the factors influencing the acute toxicity levels of parathion and chlorpyrifos. Parathion displays greater acute toxicity than chlorpyrifos, as stated above. Because of the differences in molecular weight, we have calculated the acute toxicity levels on a molar basis for a more meaningful comparison, and the acute toxicity level of parathion is about 10 fold higher than that of chlorpyrifos (45 and 440 µmoles/kg, respectively, as calculated from the acute toxicity data reported in references (1) and (2)) (13). 181 In Parameters for Pesticide QSAR and PBPK/PD Models for Human Risk Assessment; Knaak, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 1. Overview of the metabolic and toxicity relationships of phosphorothionate insecticides.

Target Acetylcholinesterase Sensitivity Certainly the simplest explanation of acute toxicity level would be the inherent sensitivity of the target enzyme, AChE, where, if there were no other factors involved, the potency of the anticholinesterase would dictate how toxic the OP compound is. However, with parathion and chlorpyrifos, as well as other phosphorothionates we have studied, the inherent potency of the active anticholinesterase metabolites, the oxons, does not reflect the overall acute toxicity level of the parent insecticide (13). Chlorpyrifos-oxon is about 5 times more potent as an anticholinesterase than paraoxon, as reflected by rat brain AChE IC50 levels (4.0 and 22.5 nM, respectively). Assessing a greater level of detail on the target AChE inhibition, inhibition kinetics studies were done with rat brain homogenates, and indicated that the inhibition constant, ki, for chlorpyrifos-oxon was about an order of magnitude higher than that for paraoxon. This difference was largely from the association of the oxon with the enzyme, with the association constants, KA, about an order of magnitude higher for chlorpyrifos-oxon than for paraoxon, while the phosphorylation constants, kp, being similar between the two oxons (Table I) (14). Therefore, target enzyme sensitivity does not dictate acute toxicity levels, and, in the case of these two insecticides, actually reflect the opposite potencies to the acute toxicity levels. Metabolism of the parent insecticides and intermediate metabolites, as well as other factors, must be important determinants of the acute toxicity levels. 182 In Parameters for Pesticide QSAR and PBPK/PD Models for Human Risk Assessment; Knaak, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

Table I. Kinetics of inhibition of rat brain acetylcholinesterase by chlorpyrifos-oxon or paraoxon. Data from reference (14) ki (bimolecular rate constant), KA (association constant) and kp (phosphorylation constant) calculated from a double reciprocal plot of the kapp and a function of inhibitor concentration, as described in reference (15) ki (mM-1 min-1) Chlorpyrifos-oxon

7528.1±271.7

Paraoxon

854.7± 33.0*


KA(mM-1) Chlorpyrifos-oxon

24109±691

Paraoxon

2054± 49*

kp(min-1)

*

Chlorpyrifos-oxon

0.295±0.013

Paraoxon

0.396±0.027

Difference between compounds, P

Comparison of Esterase Sensitivity, Metabolic Efficiency, and Toxicity

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