Inhibition of Recombinant Human Carboxylesterase 1 and 2 and Monoacylglycerol Lipase by Chlorpyrifos Oxon, Paraoxon and Methyl Paraoxon

Abstract

Oxons are the bioactivated metabolites of organophosphorus insecticides formed via cytochrome P450 monooxygenase-catalyzed desulfuration of the parent compound. Oxons react covalently with the active site serine residue of serine hydrolases, thereby inactivating the enzyme. A number of serine hydrolases other than acetylcholinesterase, the canonical target of oxons, have been reported to react with and be inhibited by oxons. These off-target serine hydrolases include carboxylesterase 1 (CES1), CES2, and monoacylglycerol lipase. Carboxylesterases (CES, EC 3.1.1.1) metabolize a number of xenobiotic and endobiotic compounds containing ester, amide, and thioester bonds and are important in the metabolism of many pharmaceuticals. Monoglyceride lipase (MGL, EC 3.1.1.23) hydrolyzes monoglycerides including the endocannabinoid, 2-arachidonoylglycerol (2-AG). The physiological consequences and toxicity related to the inhibition of off-target serine hydrolases by oxons due to chronic, low level environmental exposures are poorly understood. Here, we determined the potency of inhibition (IC₅₀ values; 15 min preincubation, enzyme and inhibitor) of recombinant CES1, CES2, and MGL by chlorpyrifos oxon, paraoxon and methyl paraoxon. The order of potency for these three oxons with CES1, CES2, and MGL was chlorpyrifos oxon > paraoxon > methyl paraoxon, although the difference in potency for chlorpyrifos oxon with CES1 and CES2 did not reach statistical significance. We also determined the bimolecular rate constants (k_inact/K_I) for the covalent reaction of chlorpyrifos oxon, paraoxon and methyl paraoxon with CES1 and CES2. Consistent with the results for the IC₅₀ values, the order of reactivity for each of the three oxons with CES1 and CES2 was chlorpyrifos oxon > paraoxon > methyl paraoxon. The bimolecular rate constant for the reaction of chlorpyrifos oxon with MGL was also determined and was less than the values determined for chlorpyrifos oxon with CES1 and CES2 respectively. Together, the results define the kinetics of inhibition of three important hydrolytic enzymes by activated metabolites of widely used agrochemicals.

Introduction

Organophosphorus (OP) pesticides have been used extensively since the 1960s for agricultural and domestic purposes. Although their use has declined since the development of the synthetic pyrethroids, they are still used in significant amounts. As recently as 2001, the EPA estimated 73 million pounds of OP insecticides were used in the United States (Kiely et al., 2004). At least one of the dialkyl phosphate metabolites (DAP) of OP insecticides was found in 50% of the United States general population in urine samples collected between 2003–2004 (http://www.cdc.gov/exposurereport/pdf/FourthReport.pdf ). The presence of DAP in urine indicates recent exposure to an OP insecticide. Children between 6–11 years of age had significantly higher levels of DAP than did adults and adolescents (Barr et al., 2004). Young children are more likely to be exposed to OP insecticides because they eat, drink and breathe more per unit of body weight than do adults and adolescents (National Research Council (U.S.) Committee on Pesticides in the Diets of Infants and Children., 1993). In addition, prenatal exposure to OP insecticides has been associated with abnormal primitive reflexes (Engel et al., 2007) and possibly with mental development (Eskenazi et al., 2007). These findings emphasize the importance of accurately modeling OP insecticide metabolism post exposure in order to aid in risk assessment.

Organophosphorus oxons, the active metabolites of many OP insecticides, exert their acute toxicity by inhibiting acetylcholinesterase via phosphorylation of the serine residue in the catalytic site (reviewed by Aldridge, 1996; Ebichon, 1996; Mileson et al., 1998). These oxons also react with other off-target proteins, many of which are members of the serine hydrolase superfamily (Aldridge, 1953; Maxwell, 1992). In fact, the covalent reaction of oxons with CES1, a serine hydrolase found in large quantities in the human liver, is one mechanism by which these compounds are detoxified and removed (Maxwell, 1992). Because CES1 is predominantly expressed in human liver, it metabolizes a range of xenobiotics containing ester, amide, and thioester bonds, e.g. ester prodrugs. CES1 is also one of several enzymes that is proposed to be responsible for the neutral cholesteryl ester hydrolase activity in macrophages (reviwed by Ghosh et al., 2010), which regulates the liberation of free cholesterol from cholesteryl esters stored in cytoplasmic lipid droplets for eventual transport to the liver and subsequent excretion (reverse cholesterol transport). Furthermore, we recently showed that CES1 regulates the amounts of the endocannabinoid 2-arachidonoylglycerol and prostaglandin glyceryl esters produced by THP1 cells (Xie et al., 2010).

Besides CES1, other serine hydrolases, including carboxylesterase 2 (CES2) and monoglyceride lipase (MGL), are also potential off targets for organophosphorus oxons. CES1, CES2, and MGL were recently dubbed “metabolic serine hydrolases” (reviewed in Simon and Cravatt, 2010). Metabolic serine hydrolases indicate enzymes that hydrolyze signaling molecules (e.g., endocannabinoids), energy storage molecules (e.g., triacylglycerols), and precursors of membrane structural components (e.g., cholesteryl esters). Products of these hydrolytic reactions include fatty acids, which are a rich fuel source. CES2 is predominantly found in the small intestine, liver, and kidney (reviewed by Satoh and Hosokawa, 2006), and has 46.8% amino acid sequence identity with CES1 (Schwer et al., 1997). CES2 can activate the anticancer drug CPT-11 (Khanna et al., 2000), but its endogenous substrates are presently unknown. MGL is primarily expressed in adipose tissue where it hydrolyzes monoglycerides, thereby freeing fatty acids for use as a source of energy, and in brain where it hydrolyzes and inactivates 2-arachidonoylglycerol (reviewed in Saario and Laitinen, 2007). The physiological consequences and toxicity, if any, related to the inhibition of these off-target serine hydrolases due to low level environmental exposures is not well understood (Carr et al., 2011). A first step toward investigating this possibility is a thorough study of the kinetics of organophosphorus oxon reactions with purified serine hydrolases. In addition, defining the reaction kinetics of organophosphorus oxons with off-target serine hydrolases could increase the accuracy of physiologically based pharmacokinetic/dynamic models that are used to predict the toxic effects of these compounds in humans.

Here, we determined the potency of inhibition (IC₅₀ values, 15 min preincubation) for chlorpyrifos oxon, paraoxon, and methyl paraoxon, the oxons derived from chlorpyrifos, parathion, and methyl parathion, three commonly used pesticides, with pure CES1, CES2, and MGL proteins in vitro. In addition, we determined the bimolecular rate constants for the reactions of chlorpyrifos oxon, paraoxon, and methyl paraoxon with CES1 and CES2, and the bimolecular rate constant for the reaction of chlorpyrifos oxon with MGL.

Materials and Methods

Chemicals and Reagents

Human recombinant CES1 and CES2 proteins were obtained by expression in baculovirus-infected Spodoptera frugiperda cells and purified as previously described (Morton and Potter, 2000). Human recombinant MGL was purchased from Cayman Chemical (Ann Arbor, MI). Rat hydrolase A was purified from adult male Sprague-Dawley rat liver as described previously (Ross et al., 2006). Chlorpyrifos oxon, paraoxon, and methyl paraoxon were all kind gifts from Dr. Howard Chambers, Department of Entomology, Mississippi State University. The oxons were of greater than 99% purity when assessed by thin-layer chromatography (Chambers et al., 1990). para-Nitrophenyl valerate (pNPV) and all other reagents and buffers were purchased from Sigma (St. Louis, MO).

Enzyme Assays

Hydrolysis reactions were performed at 37°C in a 96-well plate format in a total volume of 300 µL in 50 mM Tris-HCl (which had been adjusted to pH 7.4 at room temperature). CES1 and CES2 were diluted to final concentrations between 0.5–0.75 nM in the reaction mixtures. MGL was diluted to a final concentration of 5.5 nM in the reaction mixtures. The oxons were diluted in ethanol and added to the reaction mixture to give the desired concentrations. The final volume of ethanol in the wells was 1.5% (v/v) with CES1 and MGL, and 0.6% (v/v) with CES2. This amount of ethanol had no effect on enzymatic activity for each of the three enzymes. All reactions were corrected for nonenzymatic hydrolysis of pNPV. Nonenzymatic hydrolysis of pNPV was typically < 5% of enzymatic activity. For the IC₅₀ measurements, the enzyme and inhibitor were incubated at 37°C for 15 minutes, followed by addition of pNPV to a final concentration of 500 µM. The reaction progress was monitored by measuring the absorbance at 405 nm for 5 minutes to estimate the rate of formation of para-nitrophenol. The slopes were determined and used to calculate the enzymatic activity. The curves were linear during the 5 minute reaction period. IC₅₀ values were determined by plotting the fractional inhibition versus the concentration of oxon. Fractional inhibition was defined as: (the rate of the reaction with no oxon – the rate of the reaction with oxon) / the rate of the reaction with no oxon. IC₅₀ values were interpolated from the curve.

Kinetic Studies

The competitive kinetic scheme describing the covalent inhibition of serine hydrolases (E) by organophosphorus oxons (I) in the presence of ester substrate (S) is shown in Figure 1A. To determine the bimolecular rate constants of enzyme inactivation, an oxon (various concentrations) and pNPV (500µM) were added to the reaction buffer and brought to 37°C (5 min). The enzyme was then added to initiate the reaction. The progress of the reaction was followed by measuring the absorbance at 405 nm for either 15 minutes or 45 minutes, depending on the combination of the enzyme and oxon used. The reaction curves were fit to the equation:

$$A_{\mathrm{t}}=A_0+(A_{\mathrm{\infty }}-A_0)(1-{\mathrm{e}}^{-k_{\text{obs}}*t})$$ (1)

using SigmaPlot 8.0, and a value for the apparent first-order rate constant of enzyme inactivation (k_obs) was determined for each oxon concentration. A₀ is absorbance at time 0, A_t is absorbance at time t, A_∞ is absorbance at time infinity, t is time in s, and k_obs is the observed rate constant in s⁻¹. k_obs was then plotted against the oxon concentration and fitted to the equation:

$$k_{\text{obs}}=(k_{\text{inact}})[\mathrm{I}]/[K_{\mathrm{I}}(1+[\mathrm{S}]/K_{\mathrm{m}})+[\mathrm{I}]]$$ (2)

where k_inact is the rate constant for the inactivation (phosphorylation) of the enzyme by the oxon, K_I is the dissociation constant for EI (enzyme·inhibitor complex; i.e., the enzyme·oxon complex), [I] is the inhibitor (oxon) concentration, [S] is the pNPV concentration, and K_m is the Michaelis constant for pNPV. If one substitutes K_I’ for K_I(1 + [S]/K_m) and assumes that K_I’ >> [I], Equation (2) simplifies to:

$$k_{\text{obs}}=(k_{\text{inact}})[\mathrm{I}]/K_{\mathrm{I}}’$$ (3)

which is a linear function, where the slope is the apparent bimolecular rate constant k_i’ = k_inact / K_I’ and K_I’ is the apparent dissociation constant for the EI complex. The plots of k_obs corrected vs [I] for each reaction was fitted to both the equation for a hyperbola and the equation for a line. For each enzyme and oxon pair studied, the data typically fit the equation of a line better than that of a hyperbola as judged by r² values. The apparent bimolecular rate constants were determined from the slopes of the lines and then the true bimolecular rate constant was calculated as follows:

$$k_{\mathrm{i}}=k_{\mathrm{i}}’(1+[\mathrm{S}]/K_{\mathrm{m}})$$ (4)

where k_i represents the true bimolecular rate constant, as defined by (Main and Dauterman, 1963). K_m values were experimentally obtained for each enzyme by determining the reaction rates with varying concentrations of pNPV substrate in the absence of inhibitor. The reaction rate vs. the concentration of pNPV was plotted and the substrate concentration giving half the maximum reaction rate was determined by non-linear regression using the Michaelis-Menten equation. The values obtained for pNPV were as follows: CES1 K_m = 136 ± 40 µM, CES2 K_m = 90 ± 12 µM, MGL K_m = 158 ± 37 µM, and rat hydrolase A K_m = 32.6 µM The K_m values for CES1 and CES2 are in reasonable agreement with those previously published (Hatfield et al., 2010).

Figure 1. Effect of substrate on the progressive inhibition of a serine hydrolase by oxon.

(A) Kinetic scheme describing the inhibition of serine hydrolases (E) by oxons (I) in the presence of an ester substrate (S). The turnover number k_cat for the ester substrate (S) is a function of the rates of acylation (k₂) and deacylation (k₃) [i.e., k_cat = k₂*k₃/(k₂+ k₃)] (Streit et al., 2008); however, these steps are not explicitly shown for simplicity. (B) Inhibition of CES2 hydrolytic activity by varying concentrations of paraoxon. The progress of the pNPV hydrolysis reaction was followed by measuring the absorbance of liberated p-nitrophenol at 405 nm every 9 s for 15 minutes.

Statistical Analysis

IC₅₀ values and bimolecular rate constants were log transformed. Values in each row and each column were compared using a one way analysis of variance with post hoc Tukey analysis for three groups of data and a Student’s t-test for two groups of data. Results are noted in the footnotes for Tables 1 and 2.

Table 1.

IC₅₀ values for Reactions of Serine Hydrolases with Oxons^*

	Serine Hydrolase, IC50 (nM)**
Oxon	Carboxylesterase 1	Carboxylesterase 2	Monoacylglycerol lipase
Chlorpyrifos Oxon	0.15 ± 0.05a	0.33 ± 0.05a	5.1 ± 3.7b
Paraoxon	0.38 ± 0.10a	6.2 ± 0.3b	120 ± 40c
Methyl Paraoxon	4.8 ± 0.2b	220 ± 50c	1600 ± 960d

^*All reactions were carried out at 37°C with enzyme and inhibitor preincubated for 15 min prior to the addition of substrate.

^**Values are means ± standard deviation of at least 3 independent experiments. All values in the same row or column with different superscripts are significantly different (p< 0.001, ANOVA and Tukey’s post-hoc test).

Table 2.

Bimolecular Rate Constants for Reactions of Serine Hydrolases with Oxons

	Serine Hydrolase, kinact/KI(M−1·s−1)*
Oxon	Carboxylesterase 1	Carboxylesterase 2	Monoacylglycerol lipase
Chlorpyrifos Oxon	2.0 (±0.50) × 107	4.4 (±2.7) × 105	1.4 (±0.37) × 104
Paraoxon	1.9 (±0.48) × 106	3.4 (±2.3) × 104	N.D.
Methyl Paraoxon	1.2 (±0.57) × 105	8.1 (±2.8) × 102	N.D.

^*Values are means ± standard deviation of at least 6 independent experiments. All values have p< 0.001 when compared with values in the same row or column. N.D., not determined (insufficient inhibition to accurately measure).

Results

Inhibition Potency: IC₅₀ Measurements

IC₅₀ values were determined for chlorpyrifos oxon, paraoxon and methyl paraoxon with CES1, CES2, and MGL by preincubating each enzyme with inhibitor for 15 min at 37°C (Table 1). In general, the potency of inhibition was chlorpyrifos oxon > paraoxon > methyl paraoxon for CES1, CES2, and MGL, and IC₅₀ values were significantly different (p< 0.001) for all pair wise comparisons for each oxon with the respective enzyme (i.e., comparing values within a column of Table 1). One exception was found when comparing the inhibition of CES1 by chlorpyrifos oxon and paraoxon (p=0.209). Likewise, for each of the three oxons studied, the rank order of inhibition of the three enzymes was CES1 > CES2 > MGL, and all pair wise comparisons for each enzyme with the respective oxon (i.e., comparing values in each row of Table 1) was significantly different except when comparing the inhibition of CES1 and CES2 by chlorpyrifos oxon (p=0.161).

Determination of the Bimolecular Rate Constants (k_inact/K_I)

The bimolecular rate constant was determined for the reaction of the three oxons with the three enzymes, as described in detail in the Materials and Methods. As a representative example, the determination of the bimolecular rate constant for CES2 and paraoxon is shown in Figure 1B, supplemental Table 1, and supplemental Figure 1. Various concentrations of paraoxon were mixed with pNPV (final concentration 500 µM) in buffer and warmed to 37°C. Recombinant CES2 was added to initiate the reaction. The progress of the reaction at each concentration of paraoxon was followed by determining the absorbance at 405 nm at 9 s intervals for 15 minutes (Figure 1B). Each progress curve was then fit to an equation that describes an exponential rise to maximum (Equation 1) to obtain the apparent first-order rate constant k_obs. The value for k_obs at each paraoxon concentration was corrected by subtracting the k_obs determined in the absence of paraoxon (which represents inactivation of the enzyme not caused by the oxon inhibitor), thus generating the k_obs (corrected) values shown in supplemental Table 1. The values for k_obs (corrected) were then plotted versus each concentration of paraoxon and fit to the equation of a straight line (supplemental Figure 1, r²= 0.991), where the slope is the apparent bimolecular rate constant (k_i’= k_inact/K_I’). For this particular experiment, the apparent bimolecular rate constant was 4.4 × 10³ M⁻¹·s⁻¹. Using Equation (4), the true bimolecular rate constant was then calculated to be 2.9 × 10⁴ M⁻¹·s⁻¹. The true bimolecular rate constants for each enzyme and oxon are reported in Table 2. MGL reacts too slowly with paraoxon and methyl paraoxon to accurately determine the bimolecular rate constants. Each value in a column was significantly different than the other two values in the same column. Likewise, each value in a row was significantly different than the other values in the same row. In general, for all the enzyme-oxon pairs, the plots of k_obs (corrected) against oxon concentrations fit the equation of a straight line better than that of a hyperbola. The bimolecular rate constants for each enzyme-oxon pair were then compared to their corresponding IC₅₀ values. The two parameters exhibited a linear relationship with r²=0.8960 (plot not shown).

The bimolecular rate constant for rat hydrolase A, purified from rat liver, and chlorpyrifos oxon was also determined to be 3.3 × 10⁶ M⁻¹·s⁻¹, which lies in the middle of the range of the values found for CES1 and chlorpyrifos oxon and CES2 and chlorpyrifos oxon (Table 2).

Discussion

Accurate modeling of OP insecticide metabolism in vivo requires an understanding of the interaction of the OPs and their metabolites with the enzymes responsible for their metabolism and/or detoxication. Here, we determined the potency of inhibition (IC₅₀ values) of the liver carboxylesterases CES1 and CES2, as well as MGL, by chlorpyrifos oxon, paraoxon, and methyl paraoxon. IC₅₀ values are defined functionally and vary amongst different laboratories. We routinely preincubate the enzyme and oxon for 15 minutes at 37°C before measuring the enzyme activity. We were able to measure an IC₅₀ for each enzyme-oxon pair. In general, we found that chlorpyrifos oxon was the most effective inhibitor of the enzymes tested and that CES1 was the most reactive enzyme (Table 1). Paraoxon was noted to be a more potent inhibitor than was methyl paraoxon for each of the three enzymes (Table 1). This difference in potency is likely due to the greater reactivity of paraoxon with the enzymes as the fold difference in IC₅₀ values was almost identical to the reciprocal of the fold difference in the bimolecular rate constants for the enzyme-oxon pairs for which bimolecular rate constants could be determined (Table 2). Previous reported values for the IC₅₀ of chlorpyrifos oxon and paraoxon for the inhibition of porcine liver carboxylesterase were 2 and 3 nM, respectively (Quistad and Casida, 2000). These values are approximately one order of magnitude larger than the corresponding values we determined for CES1, which is the carboxylesterase present in the largest amount in human liver. This suggests that CES1 is more sensitive to inhibition by chlorpyrifos oxon and paraoxon than are porcine carboxylesterases. However, we do note that our incubations of enzyme and oxon were done at 37°C versus the 25°C used by Quistad and Casida (2000), which may account for some of the differences observed.

Using an approach similar to that described by Main and Dauterman (1963), we also determined the bimolecular rate constants for the reaction of chlorpyrifos oxon, paraoxon, and methyl paraoxon with CES1 and CES2 and for chlorpyrifos oxon with MGL. For both CES1 and CES2, the rank order of oxon reactivity was chlorpyrifos oxon > paraoxon > methyl paraoxon (Table 2). For chlorpyrifos oxon, the rank order of enzyme reactivity was CES1 > CES2 > MGL. The rate of reaction of MGL with paraoxon and methyl paraoxon in the presence of the substrate pNPV could not be accurately measured because the reactions were too slow to yield useful data. We also attempted to determine the two components of the bimolecular rate constant (actually referred to as the bimolecular reaction constant by Main (1964)), namely k_inact and K_I, but were unable to do so because the plots of k_obs(corrected) versus [I] were linear, implying that K_I’ was >> than [I]. Attempts to increase the concentration of the oxons so that K_I’ was not >> than [I] (see Materials and Methods) resulted in enzyme inhibition that occurred so rapidly that we were unable to capture the curvilinear portion of the exponential rise to maximum function. In general, we found good correlation between the bimolecular rate constants we were able to measure and the corresponding IC₅₀ values for the enzyme-oxon pairs.

The carboxylesterases are localized in the endoplasmic reticulum (ER) of cells (Robbi and Beaufay, 1991). They are not integral membrane proteins, rather they are found within the ER lumen tethered to the inner leaflet of the ER membrane via an integral KDEL receptor that interacts with the HIEL tetrapeptide on the C-terminus of CES proteins. It is currently unclear whether the lipid membrane affects CES kinetics in any significant manner since the active site of the enzyme is not buried within the lipid bilayer, although data from our laboratory showed that free arachidonic acid and 27-hydroxycholesterol, which are likely embedded in the membrane environment, can inhibit CES1 activity of recombinant protein and CES1 activity within intact living cells (Crow et al., 2010). Moreover, we also recently showed that the lipid peroxidation product, 4-hydroxynonenal, can inhibit CES1 activity in vitro (Borazjani et al., 2011). Therefore, in cells, it is possible that components of the ER membrane (e.g., fatty acids, oxysterols, and reactive aldehydes) might modulate the activity of the CES1 active site toward inhibitors and substrates.

To our knowledge, the bimolecular rate constants for the reaction of human CES1 and CES2 with chlorpyrifos oxon, paraoxon, and methyl paraoxon, and MGL with chlorpyrifos oxon, have not been reported. Thus, we present here for the first time the determination of the bimolecular rate constants for the reaction of pure recombinant human CES1, CES2, and MGL with these oxons. The bimolecular rates of inhibition for CES1 and pesticide oxons reported here are between one and four orders of magnitude higher than those seen for CES1 and nerve agent analogs (sarin, soman, and cyclosarin) (Hemmert et al., 2010). Therefore, CES1 appears to be very efficient at quenching pesticide oxons and is likely a critical enzyme that protects against OP poisoning in humans. A previous study (Timchalk et al., 2002) used a physiologically based pharmacokinetic/ pharmacodynamic model (PBPK/PD) to estimate the value of the bimolecular rate constant for the reaction of chlorpyrifos oxon and CES1 by fitting their model to data obtained from human studies. The value that they estimated was 5.56 × 10³ M⁻¹·s⁻¹, which is vastly lower than the value we determined, 2.0 × 10⁷ M⁻¹·s⁻¹. However, the human data used in the modeling did not include explicit chlorpyrifos oxon amounts raising some question about the accuracy of the bimolecular rate constants that were predicted by the PBPK/PD model. In fact, the authors noted that the bimolecular rate constants estimated by their model for the reaction of chlorpyrifos oxon with butyrlcholinesterase and acetylcholinesterase in humans (5.56 × 10⁵ M⁻¹·s⁻¹and 5.56 × 10³ M⁻¹·s⁻¹, respectively) were significantly lower than experimental values determined using recombinant human enzymes (2.78 × 10⁷ M⁻¹·s⁻¹and 1.67 × 10⁵ M⁻¹·s⁻¹) (Amitai et al., 1998). Other studies using purified enzymes (Shenouda et al., 2009) also determined much higher bimolecular rate constants for chlorpyrifos oxon with butrylcholinesterase and acetylcholinesterase (8.47 × 10⁸ M⁻¹·s⁻¹ and 3.33 × 10⁵ M⁻¹·s⁻¹ to 5.36 × 10⁶ M⁻¹·s⁻¹, respectively) than was estimated by (Timchalk et al., 2002). Presumably, the reason for these discrepancies is that the PBPK/PD models for OPs are examining the kinetics of OP metabolism by whole tissues and organs and not the kinetics of the interaction of OPs with isolated enzymes.

Recently, CES1 expression has been shown to be age dependent. Prenatal individuals and children less than 70 days old were found to have significantly lower levels of CES1 mRNA and protein than were adults (Yang et al., 2009) (Shi et al., 2011). Juvenile animals are more sensitive to the effects of acute exposure to OP insecticides than are adults ((Atterberry et al., 1997) (Benke and Murphy, 1975) (Pope et al., 1991)). The changes in sensitivity to OP insecticides in animals with age correlates with the expression of carboxylesterases (Benke and Murphy, 1975) (Karanth and Pope, 2000) (Moser et al., 1998). Age-dependent PBPK/PD models for rodents that incorporate the age dependent expression of carboxylesterases have been constructed (Timchalk et al., 2007). However, human PBPK/PD models in general have not taken the age dependent expression of carboxylesterases into account (Timchalk et al., 2002). Given the high level of reactivity of the oxons examined here with CES1, incorporating the age dependent hepatic expression of CES1 into the human PBPK/PD models for acute OP exposure would be expected to increase the accuracy of these models for predicting toxicity in prenatal individuals and young children.

A previous study (Mortensen et al., 1998) noted a significant difference in the IC₅₀ values of acetylcholinesterase in crude tissue preparations with chlorpyrifos oxon. When the acetylcholinesterase from these tissue fractions was isolated by immunoprecipitation, the purified enzyme had the same IC₅₀ value regardless of the tissue it was purified from. Mortensen et al. (1998) concluded that the differences observed in the IC₅₀ values using the crude preparations were due to the binding or hydrolysis of chlorpyrifos oxon by components of the tissue fraction other than acetylcholinesterase. Thus, it seems likely that some of the discrepancies in values determined by the PBPK/PD model versus those measured for pure enzymes may be the result of not including other B esterases such as CES2 and MGL in the model. It was also noted by Timchalk et al. (2002) that their model does not incorporate intestinal metabolism of chlorpyrifos, which is a possible pathway of its biotransformation since isoforms of cytochrome P450 are present in enterocytes. In addition, CES2 is abundantly expressed in the intestine and would likely react with oxons generated in situ and be inhibited. For PBPK/PD models to improve and OP metabolism in individual organs and tissues at the molecular level to be simulated, the rate constants for the interaction of OPs with individual enzymes will be needed. Knowledge of species differences in rate constants for these interactions should allow more accurate extrapolation of models from one species to another. The bimolecular rate constants we have determined for chlorpyrifos oxon, paraoxon, and methyl paraoxon with human CES1 and CES2 and for chlorpyrifos oxon with human MGL should prove useful in this modeling.

Highlights.

• IC₅₀ values and bimolecular rate constants (k_inact/K_I) of human recombinant CES1, CES2, and MGL proteins and chlorpyrifos oxon, paraoxon and methyl paraoxon were determined.

• The IC₅₀ values for the oxons with CES1, CES2, and MGL followed the rank order: chlorpyrifos oxon > paraoxon > methyl paraoxon.

• The order of reactivity for the oxons with CES1 and CES2 was chlorpyrifos oxon > paraoxon > methyl paraoxon

• Chlorpyrifos oxon was less reactive with MGL than with either CES1 or CES2

List of Abbreviations

2-AG

2-arachidonoylglycerol

CES1

Carboxylesterase 1

CES2

Carboxylesterase 2

DAP

dialkyl phosphate metabolites

MGL

monoglyceride lipase

pNPV

para-nitrophenyl valerate

OP

organophosphorus