Paraoxonase 1 (PON1) modulates the toxicity of mixed organophosphorus 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
References (68)
- et al.
Potentiation and neurotoxicity induced by certain organophosphates
Biochem. Pharmacol.
(1963) - et al.
Noncatalytic detoxication of six organophosphorus compounds by rat liver homogenates
Pestic. Biochem. Physiol.
(1990) - et al.
Role of detoxication pathways in acute toxicity levels of phosphorothionate insecticides in the rat
Life Sci.
(1994) - et al.
Malathion potentiation and inhibition of hydrolysis of various carboxylic esters by triorthotolyl phosphate (TOTP) in mice
Biochem. Pharmacol.
(1971) Current issues in organophosphate toxicology
Clin. Chim. Acta.
(2006)- et al.
Serum paraoxonase and its influence onparaoxon and chlorpyrifos-oxon toxicity in rats
Toxicol. Appl. Pharmacol.
(1990) - et al.
The role of paraoxonase (PON1) in the detoxication of organophosphates and its human polymorphism
Chem. Biol. Interact.
(1999) - et al.
A new and rapid colorimetric determination of acetylcholinesterase activity
Biochem. Pharmacol.
(1961) - et al.
Spectrophotometric assays fro the enzymatic hydrolysis of the active metabolites of chlorpyrifos and parathion by plasma paraoxonase/arlyesterase
Anal. Biochem.
(1989) - et al.
Human and rabbit paraoxonases: purification, cloning, sequencing, mapping and role of polymorphism in organophosphate detoxification
Chem. Biol. Interact.
(1993)
Pesticide interactions: effects of organophosphorus pesticides on the metabolism, toxicity, and persistence of selected pyrethroid insecticides
Pestic. Biochem. Physiol.
Paraoxonase protects against chlorpyrifos toxicity in mice
Toxicol. Lett.
Butyrylcholinesterase, paraoxonase, and albumin esterase, but not carboxylesterase, are present in human plasma
Biochem. Pharmacol.
A serine esterase released by human alveolar macrophages is closely related to liver microsomal carboxylesterases
J. Biol. Chem.
Malathion A and B esterases of mouse liver—III: In vivo effect of parathion and related PNP-containing insecticides on esterase inhibition and potentiation of malathion toxicity
Biochem. Pharmacol.
Paraoxonase 1 (PON1) Status and Substrate hydrolysis
Toxicol. Appl. Pharmacol.
Structure, function and regulation of carboxylesterases
Chem. Biol. Interact.
Potentiation of toxicity to insects and mice of phosphorothionates containing carboxyester and carboxyamide groups
Toxicol. Appl. Pharmacol.
Comparative inhibition of aliesterases and cholinesterase in rats fed eighteen organophosphorus insecticides
Toxicol. Appl. Pharmacol.
Pharmacokinetic and pharmacodynamic interaction for a binary mixture of chlorpyrifos and diazinon in the rat
Toxicol. Appl. Pharmacol.
Individual variability in esterase acticity and CYP1A levels in Chinook salmon (Oncorhynchus tshawytscha) exposed to esfenvalerate and chlorpyrifos
Aquat. Toxicol.
The toxicity of commercial jet oils
Environ. Res.
Pyrethroid insecticides: esterase cleavage in relation to selective toxicity
Science
Molecular basis for the polymorphic forms of human serum paraoxonase/arylesterase: glutamine or arginine at position 191, for the respective A or B allozymes
Am. J. Hum. Genet.
Tricresyl phosphates and cholinesterase
Biochem. J.
Malathion detoxication by human hepatic carboxylesterase and its inhibition by isomalathion and other pesticides
J. Biochem. Mol. Toxicol.
Biological activity of a tri-o-cresyl phosphate metabolite
Nature
Structure of biologically produced malathion monoacid
J. Agr. Food Chem.
Inhibition of transpermethrin hydrolysis in human liver fractions by chloropyrifos oxon and carbaryl
Drug Metab. Drug Inter.
Carboxylesterase inhibition as an indicator of malathion potentiation in mice
J. Pharmacol. Exp. Ther.
Investigation of multiple mechanisms for potentiation of malaoxon's anticholinesterase action by triorthotolyl phosphate
Proc. Soc. Exp. Biol. Med.
Expression of human paraoxonase (PON1) during development
Pharmacogenetics
Toxicity of chlorpyrifos and chlorpyrifos oxon in a transgenic mouse model of the human paraoxonase (PON1) Q192R polymorphism
Pharmacogenet. Genomics
J. Assess. Office Agr. Chem.
Cited by (45)
Early impairment of epigenetic pattern in neurodegeneration: Additional mechanisms behind pyrethroid toxicity
2019, Experimental GerontologyPig PON1: Expression and promoter methylation
2018, Gene ReportsCitation Excerpt :PON1 catalyzes the hydrolysis of thiolactones and some xenobuitoics such as organophosphate esters, unsaturated aliphatic esters aromatic carboxylic esters and carbamates (La Du, 1992; Davies et al., 1996; Costa et al., 2005). Paraoxonase is also one of the enzymes involved in the toxifying hydroperoxides generated form oxidized LDL (Jansen et al., 2009). The atherosclerosis related cardiovascular diseases are increasing with aging and therefore an alteration in PON1 expression and/or activity might be a concomitant effect during aging.
Nitric oxide synthase inhibitors protect against brain and liver damage caused by acute malathion intoxication
2017, Asian Pacific Journal of Tropical MedicineCitation Excerpt :In support of this notion, the ability of antioxidants to ameliorate the toxicant-induced DNA damage [57,58]. PON-1 is an enzyme of marked interest in view of its ability to modulate organophosphate toxicity [59]. The enzyme which possesses arylesterase and lactonase activities hydrolyzes the active metabolites (oxons) of a number of organophosphate insecticides and nerve agents [60].
Use of a probabilistic PBPK/PD model to calculate Data Derived Extrapolation Factors for chlorpyrifos
2017, Regulatory Toxicology and PharmacologyAcetylcholinesterase, butyrylcholinesterase and paraoxonase 1 activities in rats treated with cannabis, tramadol or both
2016, Asian Pacific Journal of Tropical Medicine
- 1
K.L.J. and T.B.C. contributed equally to the work described herein.