Effects of PCB Exposure on the Toxic Impact of Organophosphorus ...

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67, 311–321 (2002) Copyright © 2002 by the Society of Toxicology

TOXICOLOGICAL SCIENCES

Effects of PCB Exposure on the Toxic Impact of Organophosphorus Insecticides Russell L. Carr, 1 Jason R. Richardson, John A. Guarisco, Anil Kachroo, Janice E. Chambers, Terrilyn A. Couch, Godwin C. Durunna, and Edward C. Meek Center for Environmental Health Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, Mississippi 39762-6100 Received August 23, 2001; accepted January 16, 2002

Exposure to polychlorinated biphenyls (PCBs) can alter the metabolism of organophosphorus (OP) insecticides. Female rats were fed vanilla wafers containing either 4 mg/kg/day of Aroclor 1254 (PCBtreated) or safflower oil (oil-treated) for 50 days. Rats were then injected, ip, with corn oil, parathion (PⴝS), methyl parathion (MPⴝS), chlorpyrifos (CⴝS), paraoxon (PⴝO), methyl paraoxon (MPⴝO), or chlorpyrifos-oxon (CⴝO). In the livers of rats treated with PCBs but not OP compounds, there was induction of desulfuration (activation) of PⴝS, MPⴝS, and CⴝS, but dearylation (detoxication) was induced only with PⴝS and MPⴝS. Hepatic Aesterase hydrolysis of all three oxons was induced. Cholinesterase (ChE) activity was determined in the medulla-pons, hippocampus, corpus striatum, cerebral cortex, skeletal muscle, lung, and heart at 2 and 24 h post exposure. With CⴝS, PⴝS, and MPⴝS, differences in brain ChE inhibition were observed at 2 h (MPⴝS > PⴝS > CⴝS) but few differences were observed between oil- and PCB-treated rats. By 24 h, the level of brain ChE inhibition had increased with PⴝS and CⴝS but had decreased with MPⴝS. In rats exposed to PⴝS and CⴝS but not MPⴝS, ChE inhibition was lower in PCB-treated rats than in oil-treated rats. This suggests that pre-exposure to PCBs has a protective effect against the acute toxicity of PⴝS and CⴝS, but not MPⴝS. This protective effect does not appear to be related to the alteration of the metabolism of these compounds. The slower rate of ChE inhibition following PⴝS and CⴝS compared to MPⴝS suggests that the protection may be mediated by the PCB-induced increase in A-esterase activity. This protection appears to be related to the time between exposure and inhibition of ChE. With the oxons at 2 h, inhibition of ChE was substantial and no differences were present between the PCB- and oil-treated rats. Thus, the rapid rate of inhibition of ChE by the oxons does not afford time for the increase in A-esterase hydrolysis to effectively provide protection against inhibition of ChE. However, while no differences between oil- and PCB-treated rats were observed with MPⴝO by 24 h, PCB-treated rats exposed to PⴝO and CⴝO had lower ChE inhibition than did oil-treated rats with greater differences observed with PⴝO than CⴝO. Key Words: chlorpyrifos; parathion; methyl parathion; Aroclor 1254; PCB; acetylcholinesterase; metabolism; cytochrome P450; A-esterase; mixtures.

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To whom correspondence should be addressed. Fax: (662) 325-1031. E-mail: [email protected].

Organophosphorus (OP) insecticides are the most frequently used class of insecticide with approved uses for both household and agricultural applications (Racke, 1992). OP insecticides exert their toxicity by the inhibition of acetylcholinesterase (AChE), the enzyme responsible for the degradation of the cholinergic neurotransmitter acetylcholine. The inhibition of AChE results in the accumulation of acetylcholine in cholinergic synapses, leading to hyperstimulation of the cholinergic system. Two of the more common forms of OP insecticides are the phosphates and phosphorothionates, with the majority of OP insecticides being the latter. However, phosphorothionates are not potent inhibitors of cholinesterases (ChE) and must be metabolically converted by cytochrome P450 to their respective phosphates (oxons) to be toxic. The three insecticides used in this study, chlorpyrifos (O,O-diethyl O-(3,5,6-trichloropyridin-2-yl)-phosphorothioate), methyl parathion (O,O-dimethyl O-4-nitrophenyl phosphorothioate), and parathion (O,O-diethyl O-4-nitrophenyl phosphorothioate), are phosphorothionates. Chlorpyrifos (C⫽S) is an EPA class II insecticide and, until recent restrictions on household and termite usage, was one of the most widely used insecticides in the United States. It is currently approved for agricultural use. Methyl parathion (MP⫽S) is an EPA class I insecticide, which is restricted to agricultural use only. However, occurrences of illegal household application of methyl parathion have been reported (Anonymous, 1996). Parathion (P⫽S) is an EPA class I insecticide, and, although its current use is highly restricted in the United States, it has frequently been used as a prototypical OP insecticide in research models. Since these three phosphorothionates must be metabolically activated by the P450-mediated desulfuration reaction in order to be toxic (Neal, 1980), theoretically any chemical that increases the activity of cytochrome P450 could be expected to increase the activation of a phosphorothionate to a phosphate and thereby, potentially, lead to increased inhibition of ChE and increased toxicity. However, there is a concomitant P450mediated reaction, dearylation, which detoxifies the phosphorothionate. Both desulfuration and dearylation reactions occur through a common phosphooxythiiran intermediate and can be

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mediated by the same cytochrome P450 enzyme (Neal, 1980). Cytochrome P450 inducers, such as phenobarbital, have been reported to increase both the desulfuration and dearylation of the three phosphorothionate insecticides selected for study here (Alary and Brodeur, 1969; Chambers et al., 1994; Ma and Chambers, 1995; Sultatos, 1986, 1987; Sultatos et al., 1984). Pretreatment with phenobarbital actually decreases the toxicity of these phosphorothionates (Chambers et al., 1994; Sultatos, 1986; Sultatos et al., 1984, 1987). In contrast, ␤-naphthoflavone, which also induces cytochrome P450, has been reported to increase the dearylation of P⫽S (Chambers et al., 1994) and decrease its toxicity (Chambers and Chambers, 1990) but has also been reported to decrease the desulfuration and dearylation of C⫽S and increase its toxicity (Sultatos et al., 1984). Thus, the effects of cytochrome P450 inducers on OP insecticide toxicity appear to be dependent on the particular cytochrome P450 enzyme family that is induced. Many compounds are present in the environment that are capable of inducing the cytochrome P450 enzymes. However, most of these are not specific inducers of a single cytochrome P450 enzyme family. One group that falls into this category is that of the polychlorinated biphenyls (PCBs). PCBs are persistent environmental contaminants, which are highly chlorinated, highly lipophilic, and readily bioaccumulated in fatty tissues of all organisms. Although banned in 1977 in the U.S., PCBs are still detected at significant levels in the environment. The inherent quality of the PCBs, being a mixture of many congeners, makes them unlike many other inducers of specific cytochrome P450 enzymes in that they induce a wide variety of cytochrome P450 enzymes (Dragnev et al., 1994; Lubet et al., 1991). While this mixed induction may make it difficult to determine the role of specific P450 enzymes in the metabolism of the compound of interest, it is much more realistic with regard to what could be occurring environmentally. The PCBs found in the environment consist mainly of the highly chlorinated congeners of the original mixture, which are less susceptible to environmental degradation. While many mechanistic studies have either used individual congeners (Bushnell and Rice, 1999; Schantz et al., 1997) or reconstituted mixtures of various PCBs (Altmann et al., 2001; Hany et al., 1999), the use of commercial mixtures is an economical and effective approach to simulating human exposure (Roegge et al., 2000). In addition, Aroclor 1254 is one of the most highly chlorinated PCB mixtures and most closely represents the congeners that dominate the environmental samples (Hansen, 1999 as cited by Roegge et al., 2000). Our goal was to utilize Aroclor 1254 as an example of a mixed inducer of cytochrome P450 in order to investigate the effects of mixed induction of P450 on OP insecticide metabolism and toxicity. The dosage selected for study here is within range of occupational exposures (Lees et al., 1987; Maroni et al., 1981; Wolff, 1985) as described in Materials and Methods. In addition to the role of cytochrome P450 in the toxicity of OP insecticides, several esterases, mainly carboxylesterases

(aliesterases) and A-esterases, are present, which provide protective mechanisms against toxicity. Carboxylesterases are inhibited in a stoichiometric reaction by the active metabolites of the insecticide, thus inactivating significant amounts of the active metabolite (Maxwell et al., 1988). A-esterases function to catalytically destroy the active metabolites and provide protection against the toxic effects of some OPs (Walker and Mackness, 1987). There is some indication that the activity of these esterases can be altered by exposures to inducers. Treatment with phenobarbital has been reported to either have no effect (Chambers and Chambers, 1990) or increase carboxylesterase activity and provide protection against OP toxicity (Kaliste-Korhonen et al., 1990). Reportedly, A-esterase activity has also increased following phenobarbital treatment (Vitarius et al., 1995). Treatment with ␤-naphthoflavone has been reported to decrease the activity of carboxylesterases (Chambers and Chambers, 1990; Watson and Chambers, 1996; Watson et al., 1994). Thus, exposure to a PCB may alter the levels of these esterases such that their role in toxicity may be affected. This study was designed to determine what effect previous exposure to the PCB Aroclor 1254 (which induces xenobiotic metabolizing enzymes) would have on the toxicological impact of three OP insecticides, chlorpyrifos (C⫽S), methyl parathion (MP⫽S), and parathion (P⫽S) and their respective oxons, chlorpyrifos-oxon (C⫽O), paraoxon (P⫽O), and methyl paraoxon (MP⫽O). MATERIALS AND METHODS Chemicals. Analytical grade paraoxon, methyl paraoxon, chlorpyrifosoxon, and nitrophenyl valerate were synthesized by Dr. Howard Chambers (Department of Entomology and Plant Pathology, Mississippi State University) as previously described (Chambers and Chambers, 1989). Analytical grade parathion and methyl parathion were re-crystallized from a generous gift from Monsanto Company (St. Louis, MO). Analytical grade chlorpyrifos and 3,5,6-trichloro-2-pyridinol, for synthesis of chlorpyrifos-oxon, were a generous gift from DowElanco Chemical (Indianapolis, IN). The same batch of chemicals was used throughout the study. Aroclor 1254 (Lot #124-191) was obtained from AccuStandard, Inc. (New Haven, CT) and has been determined to be ⬎99% ortho-substituted PCBs by weight (Geller et al., 2001; Kodavanti et al., 1999). All other biochemicals were obtained from Sigma Chemical Company (St. Louis, MO). Treatment of rats. Female Sprague-Dawley (Crl:CD(SD)BR) rats (originally from Charles River) weighing 200 –250 g were used. The rats were housed in polypropylene cages containing ground corncob litter. Purina Lab Chow and tap water were freely available during experimentation. The room was maintained at a temperature of 22°C on a 12-h light:12-h dark cycle. Rats were fed vanilla wafers containing either 4 mg/kg/day of the PCB mixture Aroclor 1254 or the safflower oil vehicle for 50 consecutive days, for a total PCB dose of 200 mg/kg. This exposure occurred during 13% of the rat’s life span (2.5 years). Using the initial calculation of daily PCB exposure reported by Lees et al. (1987) and reported PCB contamination data (Lees et al., 1987; Maroni et al., 1981; Wolff, 1985), the calculated daily doses from exposure to PCBs from work area surfaces and from worker’s skin range from 0.0006 – 31.8 and 0.004 –5.6 mg/day, respectively. If we consider that a worker weighs an average of 70 kg and will work 5 days a week for 50 weeks for 10 years (13% of the human life span of 75 years), the total PCB dose ranges from

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PCB EFFECTS ON OP INSECTICIDE TOXICITY 0.021–1135.71 mg/kg from work area surface exposure and 0.014 –200 mg/kg from a worker’s skin. Thus our PCB dosage falls within the range of occupational exposures. On day 51, conscious animals (manually restrained) were given a single rapid ip injection of 1 ml/kg of parathion (3.5 mg/kg), paraoxon (1.0 mg/kg), methyl parathion (10 mg/kg), methyl paraoxon (1.0 mg/kg), chlorpyrifos (60 mg/kg), or chlorpyrifos-oxon (30 mg/kg) in corn oil. Controls received an equivalent amount of corn oil. Dosages were designed to be sublethal but of sufficient magnitude to yield greater than 90% inhibition of brain ChE and to elicit overt signs of intoxication. Animal procedures were approved by the Mississippi State University’s Institutional Animal Care and Use Committee. Rats were sacrificed at 2 or 24 h post-OP exposure. Brain, skeletal muscle, lung, and heart were removed from each animal. Brains were dissected on ice to obtain the hippocampus, cerebral cortex, corpus striatum, and medulla-pons. From rats not injected with the OP compounds, livers were removed, quick frozen in liquid nitrogen, stored at – 80°C, and later thawed for homogenization and preparation of microsomes. Microsomes were stored at – 80°C until assayed. All other tissues were frozen at – 80°C until assayed. Cholinesterase assays. All tissues were homogenized in ice-cold 0.05 M Tris-HCl buffer (pH 7.4). Brain regions were homogenized using a motorized Wheaton pestle and glass mortar. Peripheral tissues were homogenized using a Polytron at a setting of 6 for 1 min and filtered through glass wool. The incubation mixture in a total volume of 2 ml consisted of: 0.05 M Tris-HCl buffer (pH 7.4), substrate, and tissue. Protein determination was performed by the method of Lowry et al. (1951). For the P⫽S-, P⫽O-, C⫽S-, and C⫽O-treated tissues, ChE activity was determined using a modification (Chambers and Chambers, 1989) of Ellman et al. (1961), with 1.0 mM acetylthiocholine as the substrate and 5,5⬘-dithiobis(2-nitrobenzoic acid) as the chromogen. Final tissue concentrations were: medulla-pons ⫽ 0.6 mg/ml; hippocampus ⫽ 0.8 mg/ml; corpus striatum ⫽ 0.3 mg/ml; cerebral cortex ⫽ 2 mg/ml; skeletal muscle ⫽ 3.75 mg/ml; lung ⫽ 1.875 mg/ml; and heart ⫽ 1.5 mg/ml. All tissue homogenates were well stirred prior to sampling. Parallel incubations containing 0.01 mM eserine were used to correct for non-enzymatic hydrolysis. Specific activity was calculated as nmol of product produced/min mg protein. For the MP⫽S- and MP⫽O-treated tissues, ChE activity was determined using a continuous ChE method similar to that of Ellman et al. (1961). A continuous assay was used for methyl parathion and methyl paraoxon because preliminary experiments indicated that reactivation of ChE inhibited by dimethyl phosphates occurs after homogenization, even though tissues remained on ice. Tissues were homogenized in 0.05 M Tris-HCl buffer (pH 7.4) and final tissue concentrations were: medulla-pons ⫽ 2 mg/ml; hippocampus ⫽ 2 mg/ml; corpus striatum ⫽ 0.4 mg/ml; cerebral cortex ⫽ 2 mg/ml; skeletal muscle ⫽ 5 mg/ml; lung ⫽ 2.5 mg/ml; and heart ⫽ 2 mg/ml. A substrate: buffer:chromogen mixture was preincubated at 37°C for 5 min and the reaction was initiated with the addition of tissue immediately following its homogenization. The mixture was placed in a warmed (37°C) cuvette holder in a spectrophotometer and readings at 412 nm were taken every 3 s for 2 min to obtain the slope of the curve in AU/min from which specific activity (nmol of product produced/min mg protein) was calculated. Cytochrome P450 assays in non-OP treated rats. Liver microsomal ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-dealkylase (PROD) activities were quantified according to the method of Lake (1987). Specific activity was calculated as pmol of product produced/min mg protein. Liver microsomal P⫽S, MP⫽S, and C⫽S desulfuration was determined similar to the method of Ma and Chambers (1994) with modifications by Atterberry et al. (1997). Two substrate concentrations were used to test for activities of low Km app P450 enzymes (10 ␮M) and both low and high Km app P450 enzymes (50 ␮M) as previously observed in rat liver microsomes (Ma and Chambers, 1995). Specific activity was calculated as pmol oxon produced/ min mg protein. Liver microsomal P⫽S, MP⫽S, and C⫽S dearylation was determined similar to the method of Ma and Chambers (1994) with modifications by Atterberry et al. (1997). The substrate concentration of each compound used

was 50 ␮M. Specific activity was calculated as pmols product produced/min mg protein. Liver non-cytochrome P450-mediated detoxication assays in non-OPtreated rats. Whole liver A-esterase-mediated hydrolyses of P⫽O, MP⫽O, and C⫽O were determined by the method of Pond et al. (1995) using 5 mM substrate concentration for P⫽O and MP⫽O and 320 ␮M substrate concentration for C⫽O. Activity was determined as pmol product produced/min mg protein. Whole liver and liver microsomal carboxylesterase activities were determined by the method of Carr and Chambers (1991) using nitrophenyl valerate as the substrate. Specific activity was calculated as nmol product produced/min mg protein. Statistical analysis. For statistical analysis, biochemical data were analyzed using SAS on a personal computer and analysis of variance (General Linear Model), followed by mean separation, using the least square-means method. Statistical significance is reported for the p ⱕ 0.05 level.

RESULTS

Treatment with PCBs did not alter body weight (data not shown). For clarity, rats administered PCBs are designated as PCB-treated, and rats administered the PCB vehicle (safflower oil) are designated as oil-treated. For the insecticide treatments, rats receiving the insecticide vehicle (corn oil) are designated simply as controls. During the insecticide treatments, the control activities of ChE in the PCB and oil-treated rats were not significantly different in any brain region or peripheral tissue. Brain ChE following phosphorothionate exposures. At 2 h following phosphorothionate exposure, there were differences in the level of brain ChE inhibition among the 3 insecticides (Fig. 1). Inhibition following C⫽S exposure ranged from 0 –22%, with only the corpus striatum being observed as significantly different from control (Fig. 1A). Inhibition following P⫽S exposure ranged from 25–53%, with all four brain regions significantly different from controls (Fig. 1B). Inhibition following MP⫽S ranged from 57–90%, and all four brain regions were significantly different from controls (Fig. 1C). There were no differences in the amount of ChE inhibition between the oil- and PCB-treated rats with any compound except in the corpus striatum of the P⫽S-treated rats. At 24 h following phosphorothionate exposure, the level of ChE inhibition in both the oil- and PCB-treated rats exposed to C⫽S had increased significantly from the 2-h levels (Fig. 2A). However, while the ChE inhibition in the oil-treated rats exposed to P⫽S had increased significantly from the 2-h levels, there was no change in the level of ChE inhibition in the PCB-treated rats exposed to P⫽S with the exception of the corpus striatum, which was significantly increased (Fig. 2B). In contrast, with the exception of the medulla-pons in the oiltreated rats, the level of ChE inhibition in both the oil- and PCB-treated rats exposed to MP⫽S had decreased significantly from the 2-h levels (Fig. 2C). With the exception of the hippocampus in the MP⫽S treated rats, all brain regions with all compounds were significantly different from controls. The PCB-treated rats exposed to P⫽S and C⫽S had significantly lower levels of ChE inhibition when compared to the oil-

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FIG. 1. Inhibition of cholinesterase activity in the medulla-pons (MP), corpus striatum (CS), cerebral cortex (CC), and hippocampus (HC) in oil- and PCB-treated rats 2 h following exposure to (A) chlorpyrifos, (B) parathion, or (C) methyl parathion. Values are expressed as percent inhibition of the mean specific activity ⫾ SE. (n ⫽ 3). An asterisk above a bar indicates significantly different from controls (p ⱕ 0.05). Control-specific activities (nmol/min mg protein) for the discontinuous assay (A and B) were: MP ⫽ 75.60 ⫾ 3.61; CS ⫽ 228.12 ⫾ 8.48; CC ⫽ 49.03 ⫾ 0.23; and HC ⫽ 58.13 ⫾ 6.71. Control-specific activities (nmol/min mg protein) for the continuous assay (C) were: MP ⫽ 42.15 ⫾ 3.57; CS ⫽ 129.11 ⫾ 15.96; CC ⫽ 79.40 ⫾ 11.19; and HC ⫽ 74.57 ⫾ 4.75.

treated rats similarly exposed. However, ChE inhibition in the oil- and PCB-treated rats exposed to MP⫽S were not different.

FIG. 2. Inhibition of cholinesterase activity in the medulla-pons (MP), corpus striatum (CS), cerebral cortex (CC), and hippocampus (HC) in oil- and PCB-treated rats 24 h following exposure to (A) chlorpyrifos, (B) parathion, or (C) methyl parathion. Values are expressed as percent inhibition of the mean specific activity ⫾ SE. (n ⫽ 3). An asterisk above a bar indicates significantly different from controls (p ⱕ 0.05). An oval enclosing the asterisks above a pair of bars indicates a significant difference between oil- and PCB-treated within that tissue (p ⱕ 0.05). Arrows enclosed in an oval overlaying each bar indicates direction of change in the inhibition of ChE between 2 and 24 h in the tissue which that bar represents. A dash enclosed in an oval overlaying each bar indicates no change between 2 and 24 h post-exposure. Control specific activities (nmol/min mg protein) for the discontinuous assay (A and B) were: MP ⫽ 75.78 ⫾ 2.41; CS ⫽ 238.15 ⫾ 26.65; CC ⫽ 57.52 ⫾ 0.92; and HC ⫽ 53.29 ⫾ 0.14. Control-specific activities (nmol/min mg protein) for the continuous assay (C) were: MP ⫽ 44.34 ⫾ 3.78; CS ⫽ 131.11 ⫾ 14.85; CC ⫽ 81.50⫾12.01; and HC ⫽ 76.53 ⫾ 6.84.

Peripheral ChE following phosphorothionate exposures. At 2 h following phosphorothionate exposure, significant inhibition of ChE was present in the lung and heart but not in the skeletal muscle of rats exposed to C⫽S and MP⫽S (Figs. 3A and 3C). With P⫽S, significant inhibition of ChE was present in all 3 peripheral tissues (Fig. 3B). There were no differences in the levels of ChE inhibition between the oil- and PCBtreated rats with any compound, except in the lung of the P⫽S-treated rats. At 24 h following phosphorothionate exposure, significant inhibition of ChE was present in all 3 peripheral tissues in rats exposed to C⫽S, and this level of inhibition was greater than was present at 2 h (Fig. 4A). There was a significantly lower level of heart ChE inhibition in the PCB-treated rats exposed to C⫽S compared to the oil-treated rats. In the P⫽S-treated rats, significant inhibition of ChE was present in the skeletal muscle and lung of both oil- and PCB-treated rats (Fig. 4B). However, significant inhibition of ChE was present in the heart of oiltreated rats but not in the PCB-treated rats. In the skeletal muscle and heart, the level of ChE inhibition had increased significantly from the 2-h level in the oil-treated rats but in the PCB- treated rats, inhibition was similar to the 2-h level. In the lung, the level of ChE inhibition was similar to the 2-h level in the oil-treated rats, but in the PCB-treated rats, inhibition had decreased significantly. In the MP⫽S-exposed rats, significant

FIG. 3. Inhibition of cholinesterase activity in the skeletal muscle (SKM), lung, and heart in oil- and PCB-treated rats 2 h following exposure to (A) chlorpyrifos, (B) parathion, or (C) methyl parathion. Values are expressed as percent inhibition of the mean specific activity ⫾ SE (n ⫽ 3). An asterisk above a bar indicates a significant difference from controls (p ⱕ 0.05). Control-specific activities (nmol/min mg protein) for the discontinuous assay (A and B) were: SKM ⫽ 5.73 ⫾ 0.38; lung ⫽ 17.75 ⫾ 0.97; and heart ⫽ 33.22 ⫾ 1.84. Control-specific activities (nmol/min mg protein) for the continuous assay (C) were: SKM ⫽ 52.12 ⫾ 4.16; lung ⫽ 18.45 ⫾ 0.77; and heart ⫽ 31.98 ⫾ 2.60.

PCB EFFECTS ON OP INSECTICIDE TOXICITY

FIG. 4. Inhibition of cholinesterase activity in the skeletal muscle (SKM), lung, and heart in oil- and PCB-treated rats 24 h following exposure to (A) chlorpyrifos, (B) parathion, or (C) methyl parathion. Values are expressed as percent inhibition of the mean⫾SE (n ⫽ 3). An asterisk above a bar indicates a significant difference from controls (p ⱕ 0.05). An oval enclosing the asterisks above a pair of bars indicates a significant difference between oil and PCB treatments within that tissue (p ⱕ 0.05). Arrows enclosed in an oval overlaying each bar indicate direction of change in the inhibition of ChE between 2 and 24 h in the tissue which that bar represents. A dash enclosed in an oval overlaying each bar indicates no change between 2 and 24 h post exposure. Control-specific activities (nmol/min mg protein) for the discontinuous assay (A and B) were: SKM ⫽ 5.50 ⫾ 0.22; lung ⫽ 19.79 ⫾ 1.40; and heart ⫽ 30.21 ⫾ 2.11. Control-specific activities (nmol/min mg protein) for the continuous assay (C) were: SKM ⫽ 53.32 ⫾ 3.25; lung ⫽ 19.95 ⫾ 1.26; and heart ⫽ 32.18 ⫾ 1.59.

inhibition of ChE was present in the lung and heart but not in skeletal muscle (Fig. 4C). These levels of inhibition were similar to those at 2 h. No differences in ChE inhibition between the oil- and PCB- treated rats was detected in the rats exposed to MP⫽S. Brain ChE following oxon exposures. At 2 h following oxon exposure, significant inhibition of ChE was present in all brain regions, with C⫽O (55–76%), P⫽O (58 – 82%), and MP⫽O (43– 68%) (Figs. 5A–5C). There were no differences in the levels of ChE inhibition between the oil- and PCB-treated rats with any compound. At 24 h following oxon exposure, significant inhibition of ChE was present with C⫽O and P⫽O but not MP⫽O (Figs. 6A– 6C). With C⫽O, ChE inhibition in the oil-treated rats was similar to those at 2 h but in the PCB-treated rats, ChE inhibition had decreased significantly from the levels at 2 h in all brain regions with the exception of the medulla-pons (Fig. 6A). The PCB-treated rats exposed to C⫽O had significantly lower levels of ChE inhibition than the oil-treated rats in the hippocampus but not other regions. With P⫽O, ChE inhibition in the oil-treated rats was similar to those at 2 h but in the PCB-treated rats, ChE inhibition had decreased significantly from the levels at 2 h in all brain regions (Fig. 6B). The PCB-treated rats exposed to P⫽O had significantly lower lev-

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FIG. 5. Inhibition of cholinesterase activity in the medulla-pons (MP), corpus striatum (CS), cerebral cortex (CC), and hippocampus (HC) in oil- and PCB-treated rats 2 h following exposure to (A) chlorpyrifos-oxon, (B) paraoxon, or (C) methyl paraoxon. Values are expressed as percent inhibition of the mean specific activity ⫾ SE (n ⫽ 3). An asterisk above a bar indicates a significant difference from control (p ⱕ 0.05). Control-specific activities are the same as those cited in Figure 1.

els of ChE inhibition than the oil-treated rats in the medullapons, corpus striatum, and hippocampus but not in the cerebral cortex. With MP⫽O, ChE inhibition had decreased significantly from 2 h levels (Fig. 6C).

FIG. 6. Inhibition of cholinesterase activity in the medulla-pons (MP), corpus striatum (CS), cerebral cortex (CC), and hippocampus (HC) in oil- and PCB-treated rats 24 h following exposure to (A) chlorpyrifos-oxon, (B) paraoxon, or (C) methyl paraoxon. Values are expressed as percent inhibition of the mean ⫾ SE (n ⫽ 3). An asterisk above a bar indicates a significant difference from control (p ⱕ 0.05). An oval enclosing the asterisks above a pair of bars indicates a significant difference between oil and PCB treatment within that tissue (p ⱕ 0.05). Arrows enclosed in an oval overlaying each bar indicate direction of change in the inhibition of ChE between 2 and 24 h in the tissue which that bar represents. A dash enclosed in an oval overlaying each bar indicates no change between 2 and 24 h post-exposure. Control-specific activities are the same as those cited in Figure 2.

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FIG. 7. Inhibition of cholinesterase activity in the skeletal muscle, lung, and heart in oil- and PCB-treated rats 2 h following exposure to (A) chlorpyrifos-oxon, (B) paraoxon, or (C) methyl paraoxon. Values are expressed as percent inhibition of the mean specific activity ⫾ SE (n ⫽ 3). An asterisk above a bar indicates a significant difference from control (p ⱕ 0.05). Controlspecific activities are the same as those cited in Figure 3.

Peripheral ChE following oxon exposures. At 2 h following oxon exposure, significant inhibition of ChE was present in all three peripheral tissues with C⫽O and P⫽O in both oil- and PCB-treated rats (Figs. 7A and 7B). With MP⫽O, significant inhibition of ChE was only present in the lung of oil- and PCB-treated rats and in the hearts of the PCB-treated rats (Fig. 7C). There were no statistical differences between oil- and PCB-treated rats in any of the tissues with any compound. At 24 h following oxon exposure, significant inhibition of ChE was present in all three peripheral tissues with C⫽O and P⫽O but only in lung tissue with MP⫽O (Figs. 8A– 8C). With C⫽O, ChE inhibition was decreased significantly from levels at 2 h in skeletal muscle in both oil- and PCB-treated rats and in the lungs of PCB-treated rats (Fig. 8A). There was no change in ChE inhibition in the lungs of the oil-treated rats and in hearts of the PCB-treated rats. ChE inhibition was increased significantly from levels at 2 h in hearts of oil-treated rats. There were no differences between the level of ChE inhibition of oil- and PCB-treated rats in any tissue with C⫽O. With P⫽O, ChE inhibition was decreased significantly from that at 2 h in lungs of PCB-treated rats and was increased significantly from that at 2 h in hearts of oil-treated rats (Fig. 8B). There was no change in ChE inhibition in skeletal muscle of oil- or PCB-treated rats, in the lungs of oil-treated rats, or in hearts of PCB-treated rats. The PCB-treated rats exposed to P⫽O had significantly lower levels of ChE inhibition in the lung and heart than oil-treated rats but not in skeletal muscle. With MP⫽O, there was no change in ChE inhibition from that at 2 h and no differences between oil- and PCB-treated rats (Fig. 8C). Hepatic cytochrome P450 reactions. The treatment of rats with Aroclor 1254 for 50 days induced hepatic cytochrome

P450, as indicated by increased EROD and PROD activities above that of oil-treated rats (Table 1). The hepatic activation (desulfuration) of the phosphorothionates was also significantly induced by treatment with PCBs. Using a high substrate concentration of 50 ␮M, which would include both low and high Km app enzyme activity, desulfuration of all three insecticides was similar in the oil-treated rats (Table 1). In the PCB-treated rats, desulfuration of MP⫽S was similar to that of P⫽S and C⫽S, but desulfuration of C⫽S was significantly higher than that of P⫽S. Using a low substrate concentration of 10 ␮M, which represents the low Km app enzyme (high-affinity) activity, desulfuration activity of MP⫽S in the oil-treated rats was significantly higher than that of C⫽S and P⫽S, which were similar (Table 1). In the PCB-treated rats, desulfuration of all three insecticides was similar. For both the high and low substrate concentrations, desulfuration activity in the PCBtreated rats was significantly higher than that in the oil-treated rats for all three insecticides. Treatment of rats with Aroclor 1254 significantly induced cytochrome P450-mediated detoxication (dearylation) of P⫽S and MP⫽S but not of C⫽S (Table 1). In oil- and PCB-treated rats, the dearylation of C⫽S was significantly higher than the dearylation of P⫽S and MP⫽S, which were similar. Hepatic non-cytochrome P450 reactions. A-esterase hydrolysis of all three oxons in the PCB-treated rats was increased significantly above that of the oil-treated rats (Table 1).

FIG. 8. Inhibition of cholinesterase activity in the medulla-pons (MP), corpus striatum (CS), cerebral cortex (CC), and hippocampus (HC) in oil- and PCB-treated rats 24 h following exposure to (A) chlorpyrifos-oxon, (B) paraoxon, or (C) methyl paraoxon. Values are expressed as percent inhibition of the mean specific activity ⫾ SE (n ⫽ 3). An asterisk above a bar indicates a significant difference from control (p ⱕ 0.05). An oval enclosing the asterisks above a pair of bars indicates a significant difference between oil and PCB treatment within that tissue (p ⱕ 0.05). Arrows enclosed in an oval overlaying each bar indicates direction of change in the inhibition of ChE between 2 and 24 h in the tissue which that bar represents. A dash enclosed in an oval overlaying each bar indicates no change between 2 and 24 h post exposure. Control-specific activities are the same as those cited in Figure 4.

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TABLE 1 Activities of Various Hepatic Enzymes in Female Rats Exposed Orally to Either the Safflower Oil Vehicle or 4 mg/kg/day Aroclor 1254 for 50 Days

Activity Microsomal cytochrome P450 EROD PROD Desulfuration using low substrate concentration (10 ␮M) C⫽S P⫽S MP⫽S Desulfuration using high substrate concentration (50 ␮M) C⫽S P⫽S MP⫽S Dearylation C⫽S P⫽S MP⫽S A-esterase C⫽O P⫽O MP⫽O Carboxylesterase Whole liver Liver microsomal

Oil-treated

PCB-treated

Fold increase above oil-treated

1340.48 ⫾ 212.50 A 22.71 ⫾ 2.53 A

3595.49 ⫾ 163.50 B 59.33 ⫾ 5.29 B

2.68 2.61

115.91 ⫾ 7.25 A,b 111.86 ⫾ 4.64 A,b 140.38 ⫾ 2.81 A,a

190.88 ⫾ 41.12 B,a 218.82 ⫾ 38.25 B,a 258.79 ⫾ 52.25 B,a

1.65 1.96 1.84

197.25 ⫾ 15.25 A,a 141.21 ⫾ 7.25 A,a 199.63 ⫾ 31.92 A,a

568.31 ⫾ 85.22 B,a 283.84 ⫾ 23.65 B,b 410.91 ⫾ 104.68 B,ab

2.88 2.01 2.06

2208.57 ⫾ 522.91 A,a 146.08 ⫾ 7.27 A,b 130.85 ⫾ 13.98 A,b

2237.09 ⫾ 278.00 A,a 292.29 ⫾ 36.92 B,b 206.22 ⫾ 24.46 B,b

— 2.00 1.58

34.46 ⫾ 2.64 A,a 3.25 ⫾ 0.50 A,b 2.92 ⫾ 0.44 A,b

55.32 ⫾ 8.70 B,a 5.34 ⫾ 1.06 B,b 4.73 ⫾ 0.71 B,b

1.61 1.64 1.62

764.29 ⫾ 23.82 A 283.33 ⫾ 19.39 A

872.74 ⫾ 113.53 A 301.87 ⫾ 44.30 A

— —

Note. Values are expressed as mean ⫾ SE (n ⫽ 3). Microsomal CYP450 activity, desulfuration activity, and dearylation activity are expressed in pmol/min –1/mg protein –1; A-esterase and carboxylesterase activity are given in nmol/min mg protein. EROD, ethoxyresorufin O-deethylase; PROD, pentoxyresorufin O-dealkylase; C⫽S, chlorpyrifos; P⫽S, parathion; MP⫽S, methyl parathion; C⫽O, chlorpyrifos-oxon; P⫽O, paraoxon; MP⫽S, methyl paraoxon. A,B Capital letters indicate significant differences between oil- and PCB-treated values within each line (p ⱕ 0.05). a,b Lowercase letters indicate significant differences between insecticides or oxons within each treatment group for each parameter (p ⱕ 0.05).

In the oil-treated rats, A-esterase hydrolysis of C⫽O was significantly higher than that of P⫽O and MP⫽O. In contrast, carboxylesterase activity was not significantly affected by PCB treatment (Table 1). No differences in carboxylesterase activity between oil- and PCB-treated rats were present in either the hepatic microsomal fraction or whole liver. DISCUSSION

Previous work has reported that pre-exposure to inducers of cytochrome P450 can provide some degree of protection against the toxicity of organophosphorus insecticides (Alary and Brodeur, 1969; Ball et al., 1954; Brodeur, 1967; Chambers and Chambers, 1990; Chambers et al., 1994; Harbison, 1975; Mourelle et al., 1986; Sultatos, 1986; Sultatos and Minor, 1987; Sultatos et al., 1984; Triolo and Coon, 1966a,b; Welch and Coon, 1964). Thus, it was not surprising that at least some similar results were obtained in this study with PCBs. As stated earlier, the PCBs are a mixture of many congeners and are capable of inducing a wide variety of cytochrome P450 enzymes. In this study, we determined EROD and PROD activities as an indication of cytochrome P450 induction. Although

our method of PCB administration was different from that typically utilized in the literature, our level of induction was surprisingly lower than in previous reports (Dragnev et al., 1994; Lubet et al., 1991). However, the lot number of Aroclor 1254 used in this study is not as effective an inducer of cytochrome P450 as are other lot numbers (Burgin et al., 2001), which may partially explain the lower induction obtained. In addition, both PROD and EROD were significantly induced and, as stated earlier, our goal was to utilize Aroclor 1254 as an example of a mixed inducer of cytochrome P450 in order to investigate the effects of mixed induction of P450 on OP insecticide metabolism and toxicity. The characterization of the level of each P450 enzyme induced by the PCB exposure was not the focus of this study. Pre-exposure of mice to phenobarbital increased total cytochrome P450 levels, increased the rates of in vitro hepatic activation and detoxication, and decreased the toxicity of the OP insecticides studied here (Sultatos, 1986, 1987; Sultatos et al., 1984). However, pre-exposure of mice to ␤-naphthoflavone increased total cytochrome P450 levels but decreased the rates of in vitro hepatic activation and detoxication of C⫽S and

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increased the toxicity of C⫽S (Sultatos et al., 1984). In contrast, in rats, pretreatment with ␤-naphthoflavone decreased the toxicity of P⫽S (Chambers and Chambers, 1990) but did not change the in vitro activation, and detoxication was increased only in females (Chambers et al., 1994). In this study, pretreatment with PCBs increased the hepatic in vitro activation and detoxication of P⫽S and MP⫽S. However, it only increased the activation of C⫽S with no effects on detoxication. This differs from previous reports in mice treated with phenobarbital, which increased both reactions, and ␤-naphthoflavone, which decreased both reactions (Sultatos et al., 1984). Thus, the effects of induction on the activation and detoxication of C⫽S will vary depending on the enzyme induced and on the species tested. In addition, even though the activation and detoxication of a phosphorothionate may be mediated through the phosphooxythiiran intermediate, the reactions do not occur at the same ratio among P450 enzymes (Levi et al., 1988). Therefore, it is very possible that exposure to a mixed inducer, such as a PCB mixture, results in increased levels of the enzymes that preferentially activate C⫽S. Our OP metabolism data are somewhat in agreement with previously reported effects on the rate of activation of P⫽S and C⫽S following induction of P450 by phenobarbital (Ma and Chambers, 1995). During phenobarbital induction, the activation rate of P⫽S was significantly increased in the presence of both high and low substrate concentrations indicative of high (low-affinity) and low (high-affinity) Km app P450 enzymes, respectively. In the present study, the activation of P⫽S in PCB-treated rats was increased similarly with both low and high substrate concentrations. While we observed a significant increase in C⫽S activation in PCB-treated rats with both low and high substrate concentrations, the amount of increase in activation was much greater with the high- than with the low substrate concentration (p ⱕ 0.0001). This is similar to that reported by Ma and Chambers (1995), where C⫽S desulfuration was induced by phenobarbital only when high substrate concentrations were used. However, as stated above, the PCB exposure could have induced another cytochrome P450 enzyme or enzyme family, which efficiently activates C⫽S at low concentrations, explaining the PCB-induced C⫽S activation at low substrate concentrations observed here. Interestingly, the PCB-induced activation of MP⫽S is reversed from that of C⫽S. While the oil-treated levels of MP⫽S activation were similar regardless of substrate concentration, the amount of PCB-mediated increase in MP⫽S activation was much greater with the low- than with the high substrate concentration (p ⱕ 0.0299). This suggests that the PCB treatment has induced a cytochrome P450 enzyme that can efficiently activate MP⫽S at low concentrations. Regardless of oil treatment or PCB treatment, the ratio of detoxication to activation (ratio of dearylation/desulfuration using 50 ␮M as substrate) for P⫽S and MP⫽S was similar. The activation and detoxication of P⫽S was the same (oil ⫽ 1.035; PCB ⫽ 1.030) suggesting that these reactions proceed at

similar rates. With MP⫽S, detoxication was less than activation (oil ⫽ 0.656; PCB ⫽ 0.511) suggesting that the activation of MP⫽S was preferred over the detoxication of MP⫽S. This can be attributed to the more efficient activation of MP⫽S at low concentrations of MP⫽S. However, the preferred reaction with C⫽S is detoxication since its detoxication is significantly higher than its activation. Even though this difference was much greater in the oil-treated rats (oil ⫽ 11.193) than in the PCB-treated rats (PCB ⫽ 3.926), the amount of detoxication of C⫽S is greater than its activation (Ma and Chambers, 1995). The observed PCB-mediated protection against P⫽S and C⫽S toxicity is similar to previous studies investigating the effect of pretreatment with phenobarbital on OP toxicity (Sultatos, 1986; Sultatos et al., 1984). However, we did not observe a similar PCB-mediated protection against MP⫽S toxicity. This differs from the reported phenobarbital-mediated protection against MP⫽S toxicity (Sultatos, 1987). In addition, it appears that the PCB exposure gives greater protection against P⫽S toxicity than C⫽S toxicity. This is suggested by the higher level of ChE inhibition at 24 h in brain regions and peripheral tissues of the oil- and PCB-treated groups exposed to C⫽S as compared to that with P⫽S. In addition, we observed protection against the toxicity of P⫽O by PCB pretreatment. Similar results have been reported following pretreatment with phenobarbital (Alary and Brodeur, 1969; Vitarius et al., 1995). The only protective effects observed with C⫽O and MP⫽O were in the hippocampus at 24 h. The detoxication of P⫽O by cytochrome P450 has been reported to not occur to any great extent (Sultatos and Murphy, 1983) and the induction of the activation and detoxication of P⫽S were similar. Thus, it can be hypothesized that the PCBinduced changes in the cytochrome P450-mediated metabolism of P⫽S are not totally responsible for the protective effect since protective effects of PCB pretreatment were observed with both P⫽S and P⫽O. In addition, the carboxylesterases, which provide alternative binding sites for the oxons leading to stoichiometric detoxication, were not increased by PCB treatment so their role in mediating the protective effect from OP insecticide toxicity appears to be minimal. Similar to P⫽S, the protective effect of PCB pretreatment on C⫽S toxicity cannot be attributed to PCB-induced changes in its cytochrome P450-mediated metabolism or carboxylesterase detoxication. While the level of dearylation of C⫽S was not altered by the PCB pretreatment, the level of desulfuration of C⫽S was increased significantly. Thus, it stands to reason, if the activation is increased but the detoxication is not, C⫽S should be more toxic to the PCB-treated rats than to the oil-treated rats. However, this was not the case. With respect to the oil-treated rats, there were visible differences in the onset of brain ChE inhibition in C⫽S-, P⫽S-, and MP⫽S-treated rats as observed in Figure 1. This pattern is similar to what we have previously reported (Chambers and Carr, 1993). Several factors can be attributed to this differential in ChE inhibition at 2 h. First, the activation of MP⫽S by the

PCB EFFECTS ON OP INSECTICIDE TOXICITY

low Km app (high affinity) enzyme is greater than that of P⫽S and C⫽S. Thus, the higher ChE inhibition observed with MP⫽S treatment at 2 h may be a product of a more efficient rate of activation of MP⫽S. Second, the cytochrome P450 detoxication of C⫽S is much higher than that of P⫽S and MP⫽S resulting in lower ChE inhibition in the C⫽S treated rats at 2 h. Third, the A-esterase detoxication of C⫽O is much higher than that of P⫽O and MP⫽O, which also may have contributed to the lower inhibition of ChE at 2 h following C⫽S exposure. Fourth, the stoichiometric detoxication by the carboxylesterases in the liver and serum would be very important here as well. C⫽O is a potent inhibitor and P⫽O is a moderate inhibitor of these enzymes, while MP⫽O is a poor inhibitor (Chambers et al., 1990). The differential sensitivity of the carboxylesterases to these three compounds reflects the ChE inhibition observed at 2 h. This may be the most important component in the short term differential in ChE inhibition observed with the three compounds. Pre-exposure to PCB does not alter the amount of carboxylesterases thus no additional carboxylesterase-mediated protection for the PCB-treated animals is available. Thus, in the absence of the PCB, the cytochrome P450 metabolism, A-esterase metabolism, and carboxylesterases may all play a role in determining the pattern of effects of an OP insecticide and this pattern may differ from compound to compound. At first glance, the significantly increased detoxication of P⫽O by the A-esterases in the PCB-treated rats does not appear to directly account for the protective effect because the PCB pretreatment equally increased the A-esterase detoxication of C⫽O and MP⫽O but we did not observe a protective effect following C⫽O and MP⫽O. Furthermore, since the A-esterases have a much higher activity towards C⫽O than P⫽O and MP⫽O and have similar activity towards P⫽O and MP⫽O, we should have observed the greatest protection with C⫽O. Previous reports have also reached similar conclusions concerning the role of induced A-esterase levels in protecting against P⫽O toxicity (Vitarius et al., 1995). However, based on reported in vitro work (Tang and Chambers, 1999), the ability of A-esterases to reduce the circulating levels of an OP compound during an in vivo exposure would not be a rapid occurrence. It seems that A-esterases can effectively hydrolyze OP compounds but it takes a significant amount of time for a substantial amount of hydrolysis to occur. Taking this into consideration, the induced A-esterases may have played a role in the protection mediated by the PCB exposure. However, other factors may contribute to whether or not they are effective for each compound. The lack of protection from MP⫽S and MP⫽O toxicity may be related to a combination of: (1) rapid activation of MP⫽S to MP⫽O; (2) the low potential for carboxylesterase-mediated destruction of MP⫽O since carboxylesterases have a low affinity for MP⫽O; (3) the rapid inhibition of ChE following MP⫽S exposure as compared to inhibition following C⫽S and P⫽S; and (4) the rapid reactivation of ChE once inhibited by

319

the dimethyl phosphate MP⫽O. ChE inhibited by a dimethyl phosphate such as MP⫽O has a half-life of around 2 h, whereas the half-life of a diethyl phosphate is greater than 58 h (Eto, 1961). The lack of detoxication mechanisms, the rapid inhibition of ChE, and the rapid spontaneous recovery of ChE following by MP⫽S and MP⫽O exposure do not afford the A-esterases, which have been induced by the PCB exposure, sufficient time to contribute to the detoxication. Thus, time is an important factor in the lack of protection against MP⫽S/ MP⫽O by PCB exposure. With P⫽S and C⫽S, the process of activation and inhibition of ChE is much slower, which would allow the A-esterases sufficient time to hydrolyze the resulting oxons. Accordingly, the increased activity of the A-esterases in the PCB-treated rats effectively reduces the amount of oxon that would enter circulation and reach the brain and peripheral tissues to inhibit ChE. With P⫽O and C⫽O, the inhibition of ChE is rapid and the A-esterases would not have sufficient time to hydrolyze the oxons prior to inhibition of ChE. This inhibition of ChE would be fairly persistent because the two chemicals are diethyl phosphates but some recovery of ChE activity within the first 24 h is possible. It is possible that there could still be some P⫽O and C⫽O present in the body during the 2 and 24 h post-exposure period. In the oil-treated animals, some of the ChE activity would recover, but the P⫽O and C⫽O remaining in the body would re-inhibit the ChE thus maintaining similar levels of inhibition between 2 and 24 h. In the PCB-treated animals, the increased A-esterase activity could effectively destroy the oxons remaining in the body and thereby prevent the recovered ChE from being re-inhibited. The amount of inhibition of ChE at 24 h with both C⫽O and P⫽O was significantly lower than that at 2 h in most tissues. The greater decrease in inhibition with P⫽O than with C⫽O, as indicated by the differences between inhibition in the oil- and PCBtreated rats at 24 h, may be explained by simple mass action. The level of P⫽O injected was 1 mg/kg while the level of C⫽O injected was 30 mg/kg. Thus, there were significantly higher levels of C⫽O present and the A-esterases were not able to effectively remove sufficient quantities of C⫽O to allow the same amount of recovery of ChE activity. Overall, pre-exposure to PCBs decreases the toxic impact of diethyl OP insecticides but not dimethyl OP insecticides. Although PCBs induce cytochrome P450-mediated reactions associated with the phosphorothionates, this induction does not play a role in the PCB-mediated protection against toxicity afforded to the OP insecticides. Based on our data, it can be proposed that the PCB-mediated induction of A-esterase hydrolysis of the oxons provides some protection against the toxicity of the diethyl OP insecticides and this protection depends on the length of time between exposure and maximal inhibition of ChE. A longer period of time between exposure and maximal ChE inhibition affords the A-esterases time to effectively hydrolyze the active metabolites. This mechanism would not provide immediate protection against diethyl OP

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compounds which rapidly inhibit ChE (i.e., P⫽O or C⫽O) but allows faster recovery of inhibited ChE activity over time. However, it cannot be excluded that the exposure to PCBs does not alter some other physiological mechanism that contributes to the differential inhibition of ChE between PCB-treated rats and non-PCB-treated rats. ACKNOWLEDGMENTS The authors wish to acknowledge the excellent technical assistance of Lara Cook, Stephanie King, Alfred Moore, Donald Respess, Mariah Smith, Elizabeth Stokes, Jun Tang, Jennifer Wagner, and Susan Waldrop; the statistical expertise of Dr. Carolyn Boyle; and Howard W. Chambers, Department of Entomology and Plant Pathology, for supplying specific compounds used in the study. The research was partially supported by EPA Grant R825296. Research was also partially supported by the Mississippi Agricultural and Forestry Experiment Station (MAFES) under MAFES project #MISV-339010 and the College of Veterinary Medicine, Mississippi State University. This paper is MAFES publication #J-9910 and Center for Environmental Health Sciences publication #104.

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