Department of Toxicology, College of Pharmacy, University of Louisiana at Monroe, Monroe, Louisiana 71209
Received July 3, 2000; accepted September 11, 2000
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ABSTRACT |
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Key Words: organophosphate; age-related; chlorpyrifos oxonase; paraoxonase; PON1; detoxification; maturation; aging.
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INTRODUCTION |
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In addition to the inhibition of AChE, OPs also interact with other esterases relevant to cholinergic toxicity, i.e., carboxylesterases (CEs; EC 3.1.1.1) and A-esterases (AEs; EC 3.1.1.2) (Aldridge, 1953; Costa et al., 1999
; Fonnum and Sterri, 1981
). A variety of mammalian organs are rich in CEs and AEs but their physiological functions are unclear (Bogdanffy et al., 1987
; Chanda et al., 1997
; Heymann, 1980
; Mendoza et al., 1971
; Pond et al., 1995
; Stoops et al., 1975
). CEs and AEs have been reported to be important in the metabolism of various xenobiotics including OP anticholinesterases (Clement, 1984a
,b
; Maxwell, 1992
; Mentlein and Heymann, 1984
; Tang and Chambers, 1999
). CEs act as "molecular scavengers" by binding stoichiometrically to oxons, thereby reducing the number of molecules available for inhibiting AChE (Junge and Krisch, 1975
; Fonnum et al., 1985
; Maxwell et al., 1987
; Chambers et al., 1990
; Jokanovic et al., 1996
; Chanda et al., 1997
; Yang and Dettbarn, 1998
) whereas AEs such as chlorpyrifos oxonase and paraoxonase protect against anticholinesterase toxicity by catalytically inactivating oxons (Li et al., 1995
; Sultatos and Murphy, 1983a
,b
; Walker and Mackness, 1987
).
Several studies have reported that young rats are more sensitive than adults to acute OP pesticide exposures (Benke and Murphy, 1975; Brodeur and DuBois, 1963
; Moser and Padilla, 1998
; Pope and Chakraborti, 1992
; Pope et al., 1991
) but the basis for this differential sensitivity is unclear. Maturational differences in detoxification were correlated with age-related sensitivity to OP pesticide toxicity (Atterberry et al., 1997
; Benke and Murphy, 1975
; Mendoza and Shields, 1977
; Mortensen et al., 1996
; Moser et al., 1998
) but definitive mechanisms for higher sensitivity in young animals have not been delineated. Furthermore, mechanisms of age-related toxicity may be different for different OP toxicants (Moser, 1999
; Padilla et al., 2000
).
The relative sensitivity of aging mammals to OP anticholinesterases is largely unknown (Overstreet, 2000). Other exposure-related factors may make the elderly more sensitive to household pesticide applications. Neurodegenerative disorders involving the cholinergic system (e.g., Alzheimer's disease) are more common in aged human populations (Cooper et al., 1996
). Aged individuals may therefore be differentially sensitive to toxicants affecting cholinergic neurotransmission. There is little information on the levels of CEs and AEs and relative sensitivity to OP insecticides in aging. In the present study, we investigated age-related sensitivity to CPF and PS and the relationship between acute toxicity and levels of CEs and AEs in plasma, liver, and lung in neonatal (7 day), juvenile (21 day), adult (3 month) and aged (24 month) rats.
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MATERIALS AND METHODS |
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Animals and Treatments
Sprague-Dawley rats (7- and 21-day-old rats of both sexes, and 3- and 24-month-old males) were used throughout the experiment. Animals were housed in steel mesh cages with a 12-h:12-h light:dark illumination cycle and were allowed free access to food and water. All procedures involving animals were in accordance with protocols established in the NIH/NRC Guide and Use of Laboratory Animals and were reviewed by the Institutional Animal Care and Use committee at the University of Louisiana at Monroe. OP compounds were dissolved in peanut oil and injected subcutaneously (sc) at a volume of 1 ml/kg (PS) or 2 ml/kg (CPF). Maximum tolerated dosages (MTDs, i.e., highest dosages of OP agents that produced no cumulative lethality over a 7-day period) for neonatal and adult rats were obtained from previously published data (adults and neonates) or were determined essentially as described before (juvenile and aged rats) (Pope et al., 1991).
Tissue Preparations and Biochemical Assays
Carboxylesterase assay.
Carboxylesterase activity was determined by the method of Clement and Erhardt (1990) using p-nitrophenyl acetate as substrate. Blood was collected from the trunk into heparinized tubes and centrifuged for 2 min in a microcentrifuge, then plasma was removed and stored at 70°C until assay. Tissue (liver and lung) homogenates (neonatal = 4%; juvenile, adult and aged = 2% each, w/v) were prepared in ice-cold TrisHCl buffer (0.1 M, pH 7.8 at 25°C with 1% Triton X-100) in a tissue homogenizer (Polytron model PT 3000; 11,000 rpm, 30 s). Homogenates were subsequently centrifuged in a refrigerated centrifuge (Beckman model J2-21; 9000 x g) for 20 min at 4°C and the supernatant was used for CE assay. Appropriate amounts of tissue samples were added to buffer (0.1 M TrisHCl, pH 7.8 at 25°C, containing 2 mM EDTA) and the final volume was adjusted to 990 µl. Samples were preincubated at 37°C for 10 min and the reaction was started by adding 10 µl of 50 mM stock p-nitrophenyl acetate solution in acetone (final concentration = 0.5 mM). Change in absorbance at 405 nm was recorded after 5 min against a reagent blank containing only substrate. Carboxylesterase activity was calculated using a p-nitrophenol standard curve and expressed as nmol/min/ml plasma or mg protein. For in vitro studies, tissue samples were preincubated for 30 min at 37°C with a range of concentrations of chlorpyrifos oxon (CPO) and paraoxon (PO) (dissolved in ethanol) and the residual activity was measured as before. In some cases, -napthyl acetate was used as the substrate and carboxylesterase activity was measured essentially by the method of Ecobichon (1970). Appropriate amounts of tissue samples were added to buffer (0.1M TrisHCl, pH 7.8, containing 2 mM EDTA) and the final volume was adjusted to 1 ml. Samples were preincubated at 37°C for 10 min and the reaction was initiated by adding 125 µl of 10 mM stock
-napthyl acetate solution in acetone (final concentration = 1.1 mM). Change in absorbance at 322 nm was recorded after 10 min against a reagent blank containing only substrate. Carboxylesterase activity was calculated using an
-naphthol standard curve and expressed as nmol/min/ml plasma or mg protein.
Chlorpyrifos oxonase (CPO-ase) and paraoxonase (PO-ase) assay:.
CPO-ase and PO-ase activities were determined in plasma, liver, and lung by the methods described earlier (Mortensen et al., 1996; Pond et al., 1995
). Liver and lung microsomes were isolated for A-esterase activity as described by Sultatos and Murphy (1983b). Tissue homogenates were prepared in ice-cold TrisHCl buffer (0.1 M, pH 7.8) containing 2 mM CaCl2, and centrifuged at 9000 x g for 20 min using a Beckman J2-21 high-speed refrigerated centrifuge. The supernatant fraction was carefully transferred to clean tubes and centrifuged at 100,000 x g for 1 h in an ultracentrifuge (Beckman LE 80K). Pellets were resuspended in 1 ml of homogenizing buffer. For the microsomal A-esterase assay, samples were preincubated in a total of 990 µl TrisHCl buffer (0.1 M, pH 7.8) containing either 2 mM CaCl2 or 2 mM EDTA at 37°C for 10 min and the reaction was started by adding 10 µl chlorpyrifos oxon (1 mM final) or paraoxon (3 mM final). Change in absorbance was read at either 310 or 405 nm for chlorpyrifos oxonase or paraoxonase respectively in a Cintra-20 UV-Vis spectrophotometer, against a reagent blank containing only the substrate. Tissue protein was estimated by the method of Lowry and coworkers (1951). Enzyme activity was calculated using standard curves for 3,5,6-trichloro-2-pyridinol and p-nitrophenol for CPO-ase and PO-ase, respectively.
Data Analysis
Tissue esterase activity levels among the age groups were tested for statistical significance relative to adult levels by ANOVA (Tukey-Kramer analysis for all pairs), using the SAS software (version 3.2.2). We estimated Pearson moment-correlation coefficients between acute sensitivity (MTD) and esterase activity levels, using the JMP statistical package (JMP, 1995). In vitro sensitivity of CEs to oxons was analyzed using the GraphPad Prism software package (Motulsky et al., 19941995
). A probability level at 0.05 was considered significant.
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RESULTS |
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DISCUSSION |
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Levels of CEs and AEs in plasma, liver, and lung were all significantly correlated across age groups, with a measure of acute sensitivity to either CPF or PS, i.e., the maximum tolerated dosage (MTD). These results further suggest a role for CEs and AEs in age-related differences in sensitivity to some OP anticholinesterases. It should be noted, however, that the degree of correlation between esterase activity and sensitivity was generally higher with CPF than with PS. Lower levels of CPO-ase during early stages of postnatal maturation have been previously associated with higher sensitivity to CPF in rats (Atterberry et al., 1997; Mortensen et al., 1996
). The importance of paraoxonase in relative sensitivity to PS has been questioned, however. Both kinetic studies (Tang and Chambers, 1999
; Vitarius and Sultatos, 1994
) and recent studies evaluating paraoxon toxicity in PON1 knockout mice (Li et al., 2000
) suggest that paraoxonase has little role in the toxicity of PS, and thus presumably a minimal contribution to age-related differences in sensitivity to this pesticide. Basic mechanisms contributing to age-related differences in response to OP pesticides may therefore vary depending on the particular toxicant (Padilla et al., 2000
).
Earlier studies have reported a correlation between OP sensitivity and levels of esterases in plasma and liver (Atterberry et al., 1997; Benke and Murphy, 1975
; Mendoza, 1976
; Mortensen et al., 1996
; Moser et al., 1998
). The present findings are generally consistent with these previous studies. An increase in CEs and AEs during maturation was also noted in lung. The lungs have a relatively high level of CE activity and therefore could play a key role in OP detoxification under some conditions. Gaustad and coworkers (1991) suggested that lung CEs serve as important additional binding sites with higher dosages of some OP toxicants. A lower level of lung CE activity in younger rats could therefore be an additional contributing factor to their higher sensitivity. In contrast, levels of AEs in lung are comparatively low, suggesting that this biotransformation process may have little role in the overall detoxification process or age-related differences in sensitivity.
It should be noted that while both CE and AE activities were very low in neonatal, compared to adult, tissues, the levels of CE activity in juvenile tissues were proportionately much lower than those of AEs (Figs. 13). Together with the apparent minimal protection afforded by paraoxonase from PS (Li et al., 2000
), these findings suggest that the maturation of CEs may be the prominent metabolic factor leading to age-related differences in sensitivity.
While numerous studies have evaluated the relative sensitivity to OP pesticides during maturation, few have examined possible changes in sensitivity during aging. Aged rats were more sensitive than adults to PS toxicity (Table 1) while levels of CEs and AEs were similar in liver and lung from both age groups (Figs. 13
). In contrast, plasma CE was significantly lower (50%) in aged rats compared to adults. While high correlations (r = 0.8150.995) were noted between the MTD of CPF and endogenous levels of both CEs and AEs across the age groups, only plasma CE was highly correlated with sensitivity to PS (Table 2
). Plasma CEs have been reported to provide significant protection against toxicity of several OPs, including paraoxon (Clement et al., 1987
; Dettbarn et al., 1999
). Prior in vivo inhibition of plasma CE with selective inhibitors such as CBDP has been shown to potentiate the acute toxicity of several OP agents (Clement, 1984a
; Fonnum, et al., 1985
; Fonnum and Sterri, 1981
; Gupta and Dettbarn, 1987
; Yang and Dettbarn, 1998
). Maxwell (1992) emphasized the relative importance of plasma CE in detoxification of highly toxic OPs such as soman, sarin, and paraoxon. On the other hand, aged rats did not appear more sensitive than adults to CPF toxicity (Table 1
). The reason for this discrepancy is unclear: the apparently more substantial role of AE-mediated detoxification with CPF may minimize the importance of differences in plasma CE activities between the 2 age groups. The results suggest, however, that lower plasma CE levels contribute to the higher toxicity of PS in aged rats, and that plasma CE activity may be a prominent factor in differential sensitivity during maturation and aging, to this OP toxicant.
Veronesi and coworkers (1990) reported that repeated fenthion exposures caused more extensive neuropathological changes in rats treated at 12 months of age compared to similar exposures in younger (2-month-old) rats. Several studies evaluated the effects of the prototype OP toxicant diisopropylphosphorofluoridate (DFP) in aging rats. Aged (24-month) Fisher 344 rats were more sensitive than young adults (3 months) to the cholinergic toxicity of repeated DFP exposures (Michalek et al., 1990b; Pintor et al., 1988
). Although relatively similar levels of brain AChE inhibition and muscarinic receptor binding changes were noted in both young adult and aged Sprague Dawley rats during subacute DFP exposures, aged rats showed a slower recovery of both endpoints following cessation of dosing, particularly in the cortex (Michalek et al., 1990a
; Pintor et al., 1990
). Thus, both pharmacokinetic (e.g., lower plasma CE activity) and pharmacodynamic (e.g., differences in recovery of target enzyme activity between repeated exposures) could contribute to differential sensitivity to OP pesticides in aging.
The present study suggests that changes in CE activity during maturation and aging are important modulators of anticholinesterase sensitivity. In addition to plasma and liver, lung CEs may serve as additional detoxification sites for oxons. The apparently minimal role of endogenous paraoxonase in paraoxon metabolism and parathion toxicity (Li et al., 2000), along with substantial age-related differences in sensitivity to PS during maturation (Benke and Murphy, 1975
; Pope et al., 1991
), suggest that AEs may have lesser importance in the expression of differential sensitivity. Aside from the importance of environmental exposures to OP pesticides, the potentially higher acute sensitivity to PS in aging may be of practical concern because of the increasing therapeutic use of anticholinesterases in the treatment of age-related neurological disorders. Finally, these studies and others suggest that a correlation exists between esterase-mediated detoxification capacity and age-related acute sensitivity to some OP pesticides. Age-related differences in cholinergic neurochemistry, e.g., rates of acetylcholinesterase recovery and potential for presynaptic modulation of acetycholine release (Liu et al., 1999
; Michalek et al., 1990a
; Pintor et al., 1990
; Won et al., 2000
), are also correlated with sensitivity and may therefore play a role. Further mechanistic studies should clarify the relative importance of toxicokinetic and toxicodynamic differences in age-related sensitivity to these toxicants.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at the Department of Physiological Sciences, 264 McElroy Hall, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74078. Fax: (405) 744-0462. E-mail: pcarey{at}okstate.edu.
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