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
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: chlorpyrifos; parathion; methyl parathion; Aroclor 1254; PCB; acetylcholinesterase; metabolism; cytochrome P450; A-esterase; mixtures.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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-trichloro-pyridin-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 P450-mediated reaction, dearylation, which detoxifies the phosphorothionate. Both desulfuration and dearylation reactions occur through a common phosphooxythiiran intermediate and can be 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, ß-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 ß-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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Treatment of rats.
Female Sprague-Dawley (Crl:CD(SD)BR) rats (originally from Charles River) weighing 200250 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 0.000631.8 and 0.0045.6 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 0.0211135.71 mg/kg from work area surface exposure and 0.014200 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 Kmapp P450 enzymes (10 µM) and both low and high Kmapp P450 enzymes (50 µM) 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 µM. Specific activity was calculated as pmols product produced/min mg protein.
Liver non-cytochrome P450-mediated detoxication assays in non-OP-treated 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 µM 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 < 0.05 level.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 022%, with only the corpus striatum being observed as significantly different from control (Fig. 1A
). Inhibition following P=S exposure ranged from 2553%, with all four brain regions significantly different from controls (Fig. 1B
). Inhibition following MP=S ranged from 5790%, 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.
|
|
|
|
|
|
|
|
|
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). 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 ß-naphthoflavone increased total cytochrome P450 levels but decreased the rates of in vitro hepatic activation and detoxication of C=S and increased the toxicity of C=S (Sultatos et al., 1984
). In contrast, in rats, pretreatment with ß-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 ß-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) Kmapp 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 < 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 < 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 µM 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 PCB-induced 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 low Kmapp (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 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 PCB-treated 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 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 |
---|
![]() |
NOTES |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alary, J. G., and Brodeur, J. (1969). Studies on the mechanism of phenobarbital-induced protection against parathion in adult female rats. J. Pharmacol. Exp. Ther. 169, 159167.[ISI][Medline]
Altmann, L., Mundy, W. R., Ward, T. R., Fastabend, A., and Lilienthal, H. (2001). Developmental exposure of rats to a reconstituted PCB mixture or aroclor 1254: Effects on long-term potentiation and [3H]MK-801 binding in occipital cortex and hippocampus. Toxicol. Sci. 61, 321330.
Atterberry, T. T., Burnett, W. T., and Chambers, J. E. (1997). Age-related differences in parathion and chlorpyrifos in male rats: Target and nontarget esterase sensitivity and cytochrome P450-mediated metabolism. Toxicol. Appl. Pharmacol. 147, 411418.[ISI][Medline]
Ball, W. L., Sinclair, J. W., Crevier, M., and Kay, K. (1954). Modification of parathion's toxicity for rats by pretreatment with chlorinated hydrocarbon insecticides. Can. J. Biochem. Physiol. 32, 440445.[ISI]
Brodeur, J. (1967). Studies on the mechanism of phenobarbital-induced protection against malathion and EPN. Can. J. Physiol. Pharmacol. 45, 10611069.[ISI][Medline]
Burgin, D. E., Diliberto, J. J., Derr-Yellin, E. C., Kannan, N., Kodavanti, P. R., and Birnbaum, L. S. (2001). Differential effects of two lots of aroclor 1254 on enzyme induction, thyroid hormones, and oxidative stress. Environ. Health. Perspect. 109, 11631168.[ISI][Medline]
Bushnell, P. J., and Rice, D. C. (1999). Behavioral assessments of learning and attention in rats exposed perinatally to 3,3',4,4',5-pentachlorobiphenyl (PCB 126). Neurotoxicol. Teratol. 21, 381392.[ISI][Medline]
Carr, R. L., and Chambers, J. E. (1991). Acute effects of the organophosphate paraoxon on schedule-controlled behavior and esterase activity in rats: Dose-response relationships. Pharmacol. Biochem. Behav. 40, 929936.[ISI][Medline]
Chambers, H., Brown, B., and Chambers, J. E. (1990). Noncatalytic detoxication of six organophosphorus compounds by rat liver homogenates. Pestic. Biochem. Physiol. 36, 308315.[ISI]
Chambers, J. E., and Carr, R. L. (1993). Inhibition patterns of brain acetylcholinesterase and hepatic and plasma aliesterases following exposures to 3 phosphorothionate insecticides and their oxons in rats. Fundam. Appl. Toxicol. 21, 111119.[ISI][Medline]
Chambers, J. E., and Chambers, H. W. (1989). Short-term effects of paraoxon and atropine on schedule-controlled behavior in rats. Neurotoxicol. Teratol. 11, 427432.[ISI][Medline]
Chambers, J. E., and Chambers, H. W. (1990). Time course of inhibition of acetylcholinesterase and aliesterases following parathion and paraoxon exposures in rats. Toxicol. Appl. Pharmacol. 103, 420429.[ISI][Medline]
Chambers, J. E., Ma, T., Boone, J. S., Chambers, H. W. (1994). Role of detoxication pathways in acute toxicity levels of phosphorothionate insecticides in the rat. Life Sci. 54, 13571364.[ISI][Medline]
Dragnev, K. H., Beebe, L. E., Jones, C. R., Fox, S. D., Thomas, P. E., Nims, R. W., and Lubet, R. A. (1994). Subchronic dietary exposure to Aroclor 1254 in rats: Accumulation of PCBs in liver, blood, and adipose tissue and its relationship to induction of various hepatic drug-metabolizing enzymes. Toxicol. Appl. Pharmacol. 125, 111122.[ISI][Medline]
Ellman, G. L., Courtney, K. D., Andres, V., Jr., and Featherstone, R. M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 8895.[ISI][Medline]
Eto, E. (1961). Organophosphorus Pesticides: Organic and Biological Chemistry. CRC Press, Cleveland, OH.
Geller, A. M., Oshiro, W. M., Haykal-Coates, N., Kodavanti, P. R., and Bushnell, P. J. (2001). Gender-dependent behavioral and sensory effects of a commercial mixture of polychlorinated biphenyls (Aroclor 1254) in rats. Toxicol. Sci. 59, 268277.
Hansen, L. G. (1999). The Ortho Side of PCBs: Occurrence and Disposition, pp.205210 and 247249. Kluwer Academic Publishers, Boston, MA.
Hany, J., Lilienthal, H., Sarasin, A., Roth-Harer, A., Fastabend, A., Dunemann, L., Lichtensteiger, W., and Winneke, G. (1999). Developmental exposure of rats to a reconstituted PCB mixture or aroclor 1254: Effects on organ weights, aromatase activity, sex hormone levels, and sweet preference behavior. Toxicol. Appl. Pharmacol. 158, 231243.[ISI][Medline]
Harbison, R. D. (1975). Parathion-induced toxicity and phenobarbital-induced protection against parathion during prenatal development. Toxicol. Appl. Pharmacol. 32, 482493.[ISI][Medline]
Kaliste-Korhonen, E., Torronen, R., Ylitalo, P., and Hanninen, O. (1990). Inhibition of cholinesterases by DFP and induction of organophosphate-detoxicating enzymes in rats. Gen. Pharmacol. 21, 527533.[Medline]
Kodavanti, P. R. S., Kannan, N., Yamashita, N., Ward, T. R., Birnbaum, L. S., and Tilson, H. A. (1999). Differential effects of Aroclor 1254 mixtures with two lot numbers: Intracellular calcium buffering and protein kinase C translocation in rat brain. Toxicologist 48, 277.
Lake, B. G. (1987). Preparation of microsomal fractions. In Biochemical Toxicology (K. Snell, and B. Mullock, Eds.), pp. 202211. IRL Press, Oxford, England.
Lees, P. S., Corn, M., and Breysse, P. N. (1987). Evidence for dermal absorption as the major route of body entry during exposure of transformer maintenance and repairmen to PCBs. Am. Ind. Hyg. Assoc. J. 48, 257264.[ISI][Medline]
Levi, P. E., Hollingworth, R. M., and Hodgson, E. (1988). Differences in oxidative dearylation desulfuration of fenitrothion by cytochrome P450 isozymes and in the subsequent inhibition of monooxygenase activity. Pestic. Biochem. Physiol. 32, 224231.[ISI]
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265275.
Lubet, R. A., Jones, C. R., Stockus, D. L., Fox, S. D., and Nims, R. W. (1991). Induction of cytochrome P450 and other drug metabolizing enzymes in rat liver following dietary exposure to Aroclor 1254. Toxicol. Appl. Pharmacol. 108, 355365.[ISI][Medline]
Ma, T., and Chambers, J. E. (1994). Kinetic parameters of desulfuration and dearylation of parathion and chlorpyrifos by rat liver microsomes. Food Chem. Toxicol. 32, 763767.[ISI][Medline]
Ma, T., and Chambers, J. E. (1995). A kinetic analysis of hepatic microsomal activation of parathion and chlorpyrifos in control and phenobarbital-treated rats. J. Biochem. Toxicol. 10, 6368.[ISI][Medline]
Maroni, M., Colombi, A., Cantoni, S., Ferioli, E., and Foa, V. (1981). Occupational exposure to polychlorinated biphenyls in electrical workers: I. Environmental and blood polychlorinated biphenyls concentrations. Br. J. Ind. Med. 38, 4954.[ISI][Medline]
Maxwell, D. M., Brecht, K. M., Lenz, D. E., and O'Neill, B. L. (1988). Effect of carboxylesterase inhibition on carbamate protection against soman toxicity. J. Pharmacol. Exp. Ther. 246, 986991.[Abstract]
Mourelle, M., Giron, E., Amezcua, J. L., and Martinez-Tabche, L. (1986). Cimetidine enhances and phenobarbital decreases parathion toxicity. J. Appl. Toxicol. 6, 401404.[ISI][Medline]
Neal, R. (1980). Microsomal metabolism of thiono-sulfur compounds: Mechanisms and toxicological significance. In Reviews in Biochemical Toxicology (E. Hodgson, J. R. Bend, and R. M. Philpot, Eds.), pp. 131171. Elsevier, North Holland, New York.
Pond, A. L., Chambers, H. W., and Chambers, J. E. (1995). Organophosphate detoxication potential of various rat tissues via A-esterase and aliesterase activities. Toxicol. Lett. 78, 245252.[ISI][Medline]
Racke, K. D. (1992). Degradation of organophosphorus insecticides in environmental matrices. In Organophosphates: Chemistry, Fate, and Effects. (J. E. Chambers and P. E. Levi, Eds.), pp. 4772. Academic Press, San Diego, CA.
Roegge, C. S., Seo, B. W., Crofton, K. M., and Schantz, S. L. (2000). Gestational-lactational exposure to Aroclor 1254 impairs radial-arm maze performance in male rats. Toxicol. Sci. 57, 121130.
Schantz, S. L., Seo, B. W., Wong, P. W., and Pessah, I. N. (1997). Long-term effects of developmental exposure to 2,2',3,5',6-pentachlorobiphenyl (PCB 95) on locomotor activity, spatial learning and memory, and brain ryanodine binding. Neurotoxicology 18, 457467.[ISI][Medline]
Sultatos, L. G. (1986). The effects of phenobarbital pretreatment on the metabolism and acute toxicity of the pesticide parathion in the mouse. Toxicol. Appl. Pharmacol. 86, 105111.[ISI][Medline]
Sultatos, L. G. (1987). The role of the liver in mediating the acute toxicity of the pesticide methyl parathion in the mouse. Drug Metab. Dispos. 15, 613617.[Abstract]
Sultatos, L. G., and Minor, L. D. (1987). Metabolic activation of the pesticide azinphos-methyl by perfused mouse livers. Toxicol. Appl. Pharmacol. 90, 227234.[ISI][Medline]
Sultatos, L. G., and Murphy, S. D. (1983). Hepatic microsomal detoxification of the organophosphates paraoxon and chlorpyrifos oxon in the mouse. Drug Metab. Dispos. 11, 232238.[ISI][Medline]
Sultatos, L. G., Shao, M., and Murphy, S. D. (1984). The role of hepatic biotransformation in mediating the acute toxicity of the phosphorothionate insecticide chlorpyrifos. Toxicol. Appl. Pharmacol. 73, 6068.[ISI][Medline]
Tang, J., and Chambers, J. E. (1999). Detoxication of paraoxon by rat liver homogenate and serum carboxylesterases and A-esterases. J. Biochem. Mol. Toxicol. 13, 261268.[ISI][Medline]
Triolo, A. J., and Coon, J. M. (1966a). Toxicologic interactions of chlorinated hydrocarbon and organophosphate insecticides. J. Agric. Food Chem. 14, 549555.[ISI]
Triolo, A. J., and Coon, J. M. (1966b). The protective effect of aldrin against the toxicity of organophosphate anticholinesterases. J. Pharmacol. Exp. Ther. 154, 613623.[ISI][Medline]
Vitarius, J. A., O'Shaughnessy, J. A., and Sultatos, L. G. (1995). The effects of phenobarbital pretreatment on the metabolism and toxicity of paraoxon in the mouse. Pharmacol. Toxicol. 77, 1622.
Walker, C. H., and Mackness, M. I. (1987). A-esterases and their role in regulating the toxicity of organophosphates. Arch. Toxicol. 60, 3033.[ISI][Medline]
Watson, A. M., Chambers, H., and Chambers, J. E. (1994). An investigation of activities and paraoxon sensitivities of hepatic aliesterases in beta-naphthoflavone-treated rats. Toxicol. Lett. 71, 217225.[ISI][Medline]
Watson, A. M., and Chambers, J. E. (1996). The effect of high and low dosages of paraoxon in beta-naphthoflavone-treated rats. J. Biochem. Toxicol. 11, 263268.[Medline]
Welch, R. M., and Coon, J. M. (1964). Studies on the effect of chlorcyclizine and other drugs on the toxicity of several organophosphorus anticholinesterases. J. Pharmacol. Exp. Ther. 143, 192198.[ISI][Medline]
Wolff, M. (1985). Occupational exposure to polychlorinated biphenyls (PCBs). Environ. Health. Perspect. 60,133138.[ISI][Medline]