Paraoxonase 1 (PON1) modulates the toxicity of mixed organophosphorus compounds

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Abstract

A transgenic mouse model of the human hPON1Q192R polymorphism was used to address the role of paraoxonase (PON1) in modulating toxicity associated with exposure to mixtures of organophosphorus (OP) compounds. Chlorpyrifos oxon (CPO), diazoxon (DZO), and paraoxon (PO) are potent inhibitors of carboxylesterases (CaE). We hypothesized that a prior exposure to these OPs would increase sensitivity to malaoxon (MO), a CaE substrate, and the degree of the effect would vary among PON1 genotypes if the OP was a physiologically significant PON1 substrate in vivo. CPO and DZO are detoxified by PON1. For CPO hydrolysis, hPON1R192 has a higher catalytic efficiency than hPON1Q192. For DZO hydrolysis, the two alloforms have nearly equal catalytic efficiencies. For PO hydrolysis, the catalytic efficiency of PON1 is too low to be physiologically relevant. When wild-type mice were exposed dermally to CPO, DZO, or PO followed 4-h later by increasing doses of MO, toxicity was increased compared to mice receiving MO alone, presumably due to CaE inhibition. Potentiation of MO toxicity by CPO and DZO was greater in PON1−/− mice, which have greatly reduced capacity to detoxify CPO or DZO. Potentiation by CPO was more pronounced in hPON1Q192 mice than in hPON1R192 mice due to the decreased efficiency of hPON1Q192 for detoxifying CPO. Potentiation by DZO was similar in hPON1Q192 and hPON1R192 mice, which are equally efficient at hydrolyzing DZO. Potentiation by PO was equivalent among all four genotypes. These results indicate that PON1 status can have a major influence on CaE-mediated detoxication of OP compounds.

Introduction

Examining exposures to mixtures of insecticides is important for understanding the underlying mechanisms of organophosphorus (OP) compound toxicity and assessing aggregate risk. Numerous studies have examined additive and synergistic effects of combinations of OPs in vitro and in vivo. Early studies on OP mixtures in rodents found that exposure to malathion or malaoxon (MO), in combination with compounds that inhibit carboxylesterase (CaE), led to a potentiation of MO toxicity (Aldridge, 1954, Cook et al., 1957, DuBois, 1958, Murphy et al., 1959, Seume and O'Brien, 1960, Casida et al., 1961, Casida et al., 1963, Cohen and Murphy, 1971a, Cohen and Murphy, 1971b), even at doses below those required for biologically significant inhibition of cholinesterase activity (DuBois, 1969, Su et al., 1971).

The CaEs are members of a multigene family of enzymes that are widely distributed in the body with the highest activity levels being expressed in the liver, gastrointestinal tract, and the brain (Satoh and Hosokawa, 2006). CaE activity is also present in the plasma of rodents, but not humans (Williams et al., 1989, Li et al., 2005). Significant individual variability in levels of CaE in human liver microsomes has been reported, with activities varying from 5.3- to 44.7-fold, depending on the substrate used for measuring CaE activity (Hosokawa et al., 1995). Inter-individual variability of OP detoxication and bioactivation has also been identified in cytochrome P450s (Tang et al., 2001; reviewed in Furlong, 2007). CaEs can catalytically hydrolyze the carboxylic esters of malathion and MO (March et al., 1956, O'Brien, 1957, Cook and Yip, 1958, Chen et al., 1969). The bioactivation of malathion to its oxon analog forms a potent direct inhibitor of AChE (DuBois et al., 1953, March et al., 1956, Murphy and DuBois, 1957, O'Brien, 1957). When MO and CaE are combined in vitro, MO can act as both a substrate for hydrolysis and as an irreversible inhibitor of CaE and the rate of each reaction is affected by the other (Main and Dauterman, 1967). In addition to catalytic hydrolysis of OPs by A-esterases, noncatalytic hydrolysis occurs when these compounds stoichiometrically phosphorylate serine esterases (B-esterases) that are inhibited by OPs but do not hydrolyze them catalytically (e.g., CaEs and butyrylcholinesterase). The irreversible binding of CaEs to some OPs [chlorpyrifos oxon (CPO), diazoxon (DZO), and paraoxon (PO)] allows the CaEs to act as scavengers, leaving less OP available to inhibit AChE at the target site (Chambers et al., 1990).

The importance of the HDL-associated enzyme paraoxonase 1 (PON1) in OP detoxication has been known for some time (reviewed in Costa, 2006, Furlong, 2008). PON1 exhibits broad substrate specificity with different rates of hydrolysis and substrate affinity for specific OP compounds (Furlong et al., 1989, Davies et al., 1996, Pond et al., 1998, Li et al., 2000). PON1 has two common amino acid polymorphisms (Q192R and L55M) (Hassett et al., 1991). The Gln (Q)/Arg (R) substitution at position 192 affects catalytic efficiency toward some OP substrates (Adkins et al., 1993, Humbert et al., 1993, Davies et al., 1996, Li et al., 2000). Levels of plasma PON1 can vary tremendously, even among individuals with the same PON1 (192) genotype (Q/Q; Q/R; R/R), determined in part by polymorphisms in the promoter region of PON1 (Furlong, 2007). “PON1 status” is a term used to encompass both the Q192R polymorphism and the level of PON1 activity (Li et al., 1993, Richter and Furlong, 1999, Li et al., 2000). An individual's PON1 status can be determined using a two-substrate assay that provides both PON1Q192R functional phenotype and PON1 levels by plotting the plasma rates of DZO hydrolysis (at high salt) vs. PO hydrolysis (Li et al., 1993, Richter and Furlong, 1999, Costa et al., 1999, Jarvik et al., 2003). A recently-developed protocol for determining PON1 status makes use of the non-toxic substrates, phenyl acetate and 4-(chloromethyl)phenyl acetate (Richter et al., 2008, Richter et al., 2009).

PON1 knockout (PON1−/−) mice have no detectable liver or plasma hydrolytic activity toward PO or DZO and only very limited activity toward CPO. Accordingly, these animals exhibit dramatically increased sensitivity to the toxicity of CPO (Shih et al., 1998) and DZO (Li et al., 2000). Injection of purified PON1 increases the resistance of rats and mice to OP toxicity (Main, 1956, Costa et al., 1990, Li et al., 1995, Li et al., 2000). When PON1−/− mice were injected with purified human PON1 alloforms (hPON1R192 or hPON1Q192) to restore plasma PON1, hPON1R192 provided better protection than hPON1Q192 against CPO exposure, while both alloforms were equally effective in protecting against the toxicity of DZO (Li et al., 2000), and neither alloform provided protection against PO toxicity (Li et al., 2000). The extent of the protection provided by the hPON1Q192R alloforms against OP exposure was dependent on the catalytic efficiency of hydrolysis of the specific OP compound (Li et al., 2000). A study utilizing “humanized” PON1 transgenic mice provided further evidence of the role of PON1 in modulating OP toxicity. The genes of PON1−/− mice were replaced with either human hPON1R192 or hPON1Q192, and founders of each transgenic genotype were chosen that expressed hPON1 at equivalent levels (Cole et al., 2003). When these mice were exposed to CPO or its parent compound chlorpyrifos (CPS), the mice expressing hPON1Q192 were significantly more sensitive to CPO/CPS exposure than the mice expressing hPON1R192 (Cole et al., 2005).

The current study demonstrates that these differences in OP detoxication between the hPON1Q192 and hPON1R192 alloforms can have consequences beyond cholinesterase inhibition. In a combined or sequential exposure, the alloform differences can affect the subsequent toxicity (or efficacy) of compounds metabolized by CaE, even when the compound is not metabolized directly by PON1. We demonstrate that CPO, DZO, and PO inhibit CaE in vitro and in vivo and increase MO toxicity in vivo, and that PON1 status modulates the degree of MO potentiation by virtue of its impact on the metabolism of CPO and DZO.

Section snippets

Chemicals

Chlorpyrifos oxon (CAS 5598-15-2; 98% purity), diazoxon (diazinon-O-analog; CAS 962-58-3; 96% purity), paraoxon (O,O-diethyl-O-p-nitrophenylphosphate; CAS 311-45-5; 98.4% purity), malaoxon (CAS 1634-78-2; 88% purity) and tri-ortho-cresyl phosphate (CAS 1330-78-5; 98.5% purity) were purchased from Chem Service (West Chester, PA, USA). Acetylthiocholine, 5,5′-dithio-bis-nitrobenzoic acid (DTNB), p-nitrophenyl valerate, and phenyl acetate were from Sigma-Aldrich (St. Louis, MO). All other

Results

The first objective of this study was to determine, through the use of PON1+/+ and PON1−/− mice, whether PON1 modulates susceptibility to exposures of OP insecticide mixtures. The second objective was to determine the impact of PON1 status (hPON1Q192R genotype and plasma PON1 level) on the toxicity of OP insecticide mixtures, through the use of humanized hPON1Q192 and hPON1R192 transgenic mice. The design involved measuring inhibitions of AChE (brain and diaphragm) and CaE (liver and serum) in

Discussion

This research focused on the relative importance of the two hPON1Q192R alloforms in protecting against OP toxicity during sequential mixed OP exposures, using transgenic mouse models that included strains with different PON1 status (PON1+/+, PON1−/−, hPON1R192 and hPON1Q192). OP exposures are often multi-route and multi-chemical in nature. Based on a determination that OPs form a common mechanism group based on their shared ability to inhibit AChE in both the central and peripheral nervous

Acknowledgments

The authors express their appreciation to Drs. Diana Shih, Aldons J. Lusis, and Aaron Tward for kindly providing the PON1−/− mice and the hPON1Q192 and hPON1R192 transgenic mice used in this study, and the University of Washington Department of Biostatistics for assistance with statistical analyses. This work was supported by the National Institute of Environmental Health Sciences (NIEHS) [grant numbers P42ES04696, R01ES09883; P30ES07033, and P01ES09601], and by grant number RD83170901 from the

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    K.L.J. and T.B.C. contributed equally to the work described herein.

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