Department of Pharmacology and Cancer Biology, Duke University Medical Center, P.O. Box 3813, Durham, North Carolina 27710
Received March 3, 2000; accepted May 15, 2000
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ABSTRACT |
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Key Words: sarin; choline acetyltransferase; acetylcholinesterase; muscarinic acetylcholine receptor; nicotinic acetylcholine receptor; neurotoxicity; Gulf War.
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INTRODUCTION |
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The neurotoxicity of sarin has been evaluated in different rodent and mammalian species, and the acute toxic effects are supposed to be mediated by inhibition of acetylcholinesterase (AChE). The main clinical features associated with acute sarin intoxication are seizure, fasciculation, tremor, and hypothermia (Taylor, 1985). The appearance of these symptoms correlates with the inhibition of AChE, both in the central nervous system (CNS) and peripheral nervous system (PNS) (Gupta et al., 1991
). This is followed by excessive accumulation of acetylcholine, leading to hyperactivation of nicotinic and muscarinic acetylcholine receptors.
Excessive accumulation of acetylcholine leads to activation of ligand-gated ion channels, and of nicotinic acetylcholine receptors (nAChR), and muscarinic acetylcholine receptors (mAChR). These receptors activate diverse kinds of cellular responses by distinct signaling mechanisms (Wess, 1996). Indeed, previous studies from our laboratory and others have shown that organophosphate compounds cause differential regulation of nAChR and mAChR (Huff et al., 1994
; Katz et al., 1997
). In vitro studies by Bakry et al. (1988) suggested that sarin binds to nAChR and modulates its ligand-binding characteristics. A recent study showed a decrease in high-affinity choline uptake by the insecticide chlorpyrifos (O, O-diethyl 3,5,6-trichloropyridenyl phosphorothioate) (Liu and Pope, 1996
). The levels of acetylcholine in the CNS can be regulated by different metabolic pathways, e.g., by the inhibition or activation of AChE and choline acetyltransferase and regulation of high-affinity, sodium-dependent choline transporter (Taylor and Brown, 1999
). Rats exposed to soman and sarin have been found to have a decrease in high affinity choline uptake in cortex and hippocampus (Whalley and Shih, 1989
). From all of these studies, it is apparent that changes in acetylcholine-related metabolism are the key regulators of CNS toxicity induced by organophosphate compounds, including sarin.
Several studies in the past have reported consistent inhibition of AChE by sarin; however, its effects on choline acetyltransferase (ChAT) are shown to be variable, with some having no effect (Sivam et al., 1984) and others with inhibition of no consequence (Kobayashi et al., 1986
). Yet another study by Brookes and Goldberg (1979), using cultured spinal cord cells, showed activation of ChAT by a closely related compound, diisopropylphosphoroflouridate. Because of the central role of the cholinergic system in the manifestation of toxicity by sarin, we decided to evaluate acute effects of sarin on the interplay between the cholinergic parameters, i.e., AChE, ChAT, nAChR, and mAChR, concurrently, in a single study. Our studies show that sarin caused inhibition of plasma BChE and brain region-specific AChE and persistent activation of ChAT. They also show that sarin caused increased binding of nAChR- and mAChR-specific ligands.
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MATERIALS AND METHODS |
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Animals
Male Sprague-Dawley rats (200250g) were obtained from Zivic-Miller Laboratories (Allison Park, PA) and housed in the Duke University Medical Center vivarium on a 12-h dark-light cycle. The animals were allowed food and water ad libitum. All the treatments and procedures on the animals were carried out strictly according to the recommended guidelines by the Army and the Duke University Institutional Animal Care and Use Committee.
Treatment of Animals and Tissue Retrieval
Animals were treated with a single intramuscular injection of 100 µg/kg/ml in normal saline into the thigh muscle for the LD50 time-course study (Abou-Donia, et al., unpublished observation). For LD50 treatments, a minimum of 10 animals were used, of which 2 to 3 died within 60 min of treatment. The remaining surviving animals were sacrificed for tissue and blood collection, to carry out biochemical estimations. For dose study, sarin was diluted with normal saline to give a final concentration of 0.01, 0.1, 0.5, or 1 x LD50 to each animal. Control animals received an equal volume of vehicle. At the termination of the experiment, the animals were anesthetized with 0.2 ml of ketamine/xylocaine and blood was drawn into a heparinized syringe. Animals were dissected, and the brain was removed and washed thoroughly with ice-cold normal saline to remove blood. Brain regions (cortex, midbrain, cerebellum, and brainstem) were dissected on ice and snap frozen in liquid nitrogen.
Enzyme and Receptor Assays
Cholinesterase determination.
AChE in brain regions and BChE in plasma activities were determined according to the method of Ellman et al. (1961), modified for assay in a Molecular Devices UV Max Kinetic Microplate Reader, as previously described (Abou-Donia, et al., 1996). In brief, brain regions were weighed and 10% homogenate was prepared in 0.1 M phosphate buffer, pH 8.0, containing 0.5% Triton X-100. The homogenate was centrifuged at 5000 x g for 10 min at 4°C. The supernatant was used as the source of the enzyme. Blood samples were centrifuged at 5000 x g for 10 min to separate plasma. All samples were stored at 70°C until use. All tissue supernatants were diluted 1:10 with PBS containing 10 mM MgCl2, pH 7. Twenty µl of diluted supernatant was used for each assay in a total volume of 200 µl of buffer or 0.2 mM acetylthiocholine iodide. For plasma BChE determination, the plasma was diluted 1:10 in PBS containing 10 mM MgCl2 and assayed as described for brain regions, except that 0.2 mM butyrylthiocholine (BSCh) was used as substrate in the presence of 5 x 107 M AChE inhibitor, 1,5-bis-(N-allyl-N-N-dimethyl-4-ammoniumphenyl) pentane-3-one dibromide. The reaction was started by the addition of 0.1 mM 5,5`-dithiobis-2-nitrobenzoic acid (DTNB) in PBS. The blank contained buffer in the place of substrate, and the enzyme activity was monitored by recording the absorbance at 412 nm. Protein concentration was determined by BCA method according to Smith et al. (1985). The enzyme activities are expressed as µmol substrate hydrolyzed/min/mg protein for brain regions and nmol substrate hydrolyzed/min/mg protein for plasma.
Determination of choline acetyltransferase.
Choline acetyltransferase (ChAT) activity in brain regions was determined according to the method of Fonnum (1975). Briefly, the tissues were homogenized in 50 mM phosphate buffer, pH 7.4, containing 0.5% TritonX-100 and centrifuged at 5000 x g for 10 min at 4°C. The supernatant was used as the source of enzyme. The assay was carried out in 50 mM phosphate buffer, pH 7.4, containing 0.2 M sodium chloride, 10 mM EDTA, 100 µM eserine, 5 mM choline chloride, 200 µM acetyl CoA (0.25 µCi [3H]acetyl CoA), in a final volume of 200 µl, for 30 min at 37°C. The reaction was stopped by adding an equal volume of 1.5% tetraphenyl boron in 3-heptanone, vortexed thoroughly, and centrifuged at 5000 x g for 5 min to separate the organic phase. The acetylcholine level was determined by counting the organic phase. Enzyme activity was expressed as pmol acetylcholine formed/min/mg protein.
SDSPAGE and Western blotting of ChAT.
A suitable aliquot of 5000 x g supernatant containing 25 µg of protein was denatured with sample buffer. Proteins were separated and transferred to PVDF membranes as described by Khan et al. (1994). The membranes were incubated with 5% non-fat dry milk containing 0.5% Surfactin in Tris-buffered saline, pH 7.4, for 1 h at room temperature to block the nonspecific sites. Membranes were incubated with the primary antibody overnight at 4°C at 1:1000 dilution. The membranes were washed with 5% non-fat dry milk containing Surfactin x 3 for 15 min each, following which the incubation with secondary antibody conjugated to horseradish peroxidase was carried out for 1 h at room temperature. After extensive washing, the reaction was developed by chemiluminescence using an ECL kit supplied by Amersham Biosciences.
Nicotinic acetylcholine receptor (nAChR) binding assay.
[3H]Cytisine was used as the specific ligand for binding studies with nAChR according to the method described by Slotkin et al. (1999). The tissue was homogenized by polytron in 50 mM TrisHCl, pH 7.4, containing 120 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, and 2.5 mM MgCl2. The membranes were sedimented by centrifuging at 40,000 x g for 10 min. The resulting membrane pellet was resuspended in the same buffer, using Teflon pestle glass homogenizer in a volume sufficient to give 1.5 to 2.0 mg/ml protein. An aliquot of membrane preparation containing 200 µg protein was used to carry out the incubation with 1 nM [3H]cytisine at 4°C for 75 min. Nonspecific binding was carried out in the presence of 1 µM nicotine ditartrate. The labeled membranes were trapped on membrane filters using a rapid vacuum filtration system, and the results are expressed as specific binding (dpm)/mg protein.
Muscarinic acetylcholine receptor (mAChR) binding assay.
For the assay of mAChR, the tissue was homogenized in 10 mM phosphate buffer, pH 7.4, and centrifuged at 40,000 x g for 10 min, and the membranes were suspended in the same buffer at the protein concentration of 1.52.5 mg/ml as described by Huff et al. (1994). Muscarinic receptor in the CNS comprises a family of 5 distinct members (m1m5). We carried out ligand-binding studies with m2-mAChR, because of its central role in memory and learning, and our previous studies (Huff et al., 1994) have shown that m2-mAChr is selectively regulated by organophosphate. The m2-mAChR binding was carried out by using m2-selective ligand, [3H] AFDX 384 at room temperature for 60 min. Nonspecific binding was carried out in the presence of 2.22 µM atropine. Ligand-bound membranes were trapped on glass filters presoaked with 0.1% polyethyleneimine using rapid vacuum filtration as described for the nAChR assay. The results are expressed as specific binding (dpm)/mg protein.
Statistical analysis.
The data were analyzed by Student's t-test for statistical significance. The graphs were generated on Excel graphics for Macintosh and are presented as mean ± SE.
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RESULTS |
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Effect of Sarin on Plasma Cholinesterase Activity
Initially we carried out a time course study to evaluate the inhibitory potential of sarin after a single LD50 dose. Sarin treatment at 1 x LD50 dose resulted in 45% decrease in plasma butyrylcholinesterase activity 30 min after exposure and continued to decrease up to 5565% by 3 and 15 h (Fig. 1
). The maximum inhibitory effect persisted up to 15 h after treatment.
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DISCUSSION |
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Several studies have reported inhibition of AChE by sarin (Lim et al., 1983 and references therein). The regulatory role of sarin on biosynthesis and degradation of acetylcholine and its role on the CNS nAChR and mAChR, however, has not been reported in a single study. It is important to evaluate these aspects of the cholinergic pathway, because the interplay between each component of the cholinergic pathway would ultimately affect the neurotoxicity of sarin. The present study provides data on all of these aspects of sarin-induced toxicity.
Inhibition of plasma BChE is the hallmark of the neurotoxicity induced by a large number of organophosphates, including sarin. Our time-course study with 1 x LD50 sarin dosage shows that sarin treatment resulted in a significant decrease in BChE activity in plasma and AChE inhibition in cortex, brainstem, midbrain, and cerebellum. The dose-response study showed that significant inhibition in BChE activity is observed only at the higher dose of sarin at 15 h of treatment. However, it is likely that lower doses such as 0.01and 0.1 x LD50 may also have an inhibitory effect at earlier time periods, which is subsequently recovered. It is known that sarin-inhibited AChE ages slowly (Clement, 1982), and therefore, the inhibition might have subsided by 15 h, the time of our study. Increase in AChE activity at 0.01 x LD50 sarin may be mediated by an indirect mechanism as opposed to the inhibition of AChE by sarin, which is by direct interaction with the enzyme.
Our results also suggest that there are regional differences in the brain severity to inhibition of AChE by lower doses of sarin, with midbrain showing inhibition of AChE activity at 0.1 x LD50, whereas in the cortex and brainstem, the inhibition was observed at 0.5 x LD50. This differential response could reflect the total enzymatic activity present in each region. Thus, the threshold level of cortex and other brain regions such as striatum AChE may be higher than in the rest of the regions.
ChAT is a specific marker of cholinergic innervation in the CNS, which catalyzes the final step in the biosynthesis of acetylcholine (Wu and Hersh, 1994). Although it is believed that ChAT is not the rate-limiting enzyme in the availability of acetylcholine in the CNS, it can have a modulatory role in the cholinergic system. Indeed, in the past attempts have been made to use selective inhibitors of choline acetyltransferase as possible in vivo protection mechanisms against soman-induced neurotoxicity (Harris et al., 1982
; Schoene et al., 1977
). Sterling et al. (1988) reported that a quaternary salt of hydroxyethylnaphthylvinyl pyridine provided protection against soman-induced mortality when given 23 min prior to soman treatment. Our data on ChAT activation by sarin in cortex and brainstem prove that enhanced enzyme activity may have some consequence, at least in the early period of exposure. Similarly, Brookes and Goldberg (1979), using mouse spinal cord cell culture, found that diisopropylphosphoroflouridate (DFP) exposure caused activation of ChAT. Others found no effect on the enzyme activity in response to sarin or DFP exposure (Sivam et al., 1984
). The reasons for these differences are not known. Significant inhibition of ChAT activity in the brainstem at early time periods following LD50 sarin administration may suggest a direct inhibitory effect.
ChAT activation by sarin in vivo is interesting, because it is known that sarin-inhibited AChE is reactivated faster than other nerve agent-inhibited AChE (Clement, 1991; Clement et al., 1991
; Schoene, 1978
). Clement (1982) reported that soman-inhibited AChE ages faster than a sarin-inhibited enzyme. Therefore, in view of persistent activation of ChAT, as observed in our studies, it is reasonable to assume that even when AChE inhibition by sarin is not pronounced (because of faster reactivation), the still higher level of acetylcholine could be available at the presynaptic terminals. However, the role of vesicular acetylcholine transporter remains to be evaluated under these conditions.
ChAT activation has been observed under a variety of conditions; mostly related to trophic factors and survival (Cavicchioli et al., 1991; Fusco et al., 1989
; Li et al., 1995
; Mobley et al., 1985
; Wu and Hersh, 1994
). However, other modifications such as phosphorylation and proteolysis have also been shown to regulate the enzyme activity (Bruce and Hersh, 1989
; Wu et al., 1995
). Sarin-induced activation of ChAT activity may involve proteolytic cleavage of the enzyme. An increase in electrical activity has been shown to cause increased proteolytic activity by. Furthermore, it has been shown that cholinergic stimulation causes protease(s) activation that leads to synapse loss in activity-dependent manner (Liu et al., 1994
). Therefore, it is likely that increased ChAT activity following sarin exposure may be a consequence of protease-mediated activation of the enzyme. This view needs further studies.
Inhibition of AChE following OP poisoning causes excessive stimulation of CNS AChRs. Under acute exposure conditions, overstimulation leads to seizure and chronic activation may lead to impairment of memory function (Taylor, 1985). A critical role of nAChR and mAChR has been implicated in all these processes (McGehee, 1999
; Wess, 1996
). Our data suggest that acute sarin exposure significantly increases binding densities of respective ligands for nAChR and m2-mAChR. Earlier in vitro studies (Bakry et al., 1988
) reported inhibition in binding of high affinity m2 receptor ligand [3H]CD to mAChR by several organophosphates, including sarin. This effect was found to be selective for mAChR as the inhibition for nAChR was not pronounced. A recent study extended these observations by reporting that not only neuronal type nAChR but also electric ray nAChR, which is very similar to muscular AChR structurally and pharmacologically, binds organophosphates of diverse structures (Katz et al., 1997
). Binding of these organophosphate compounds to the nAChR is believed to be at a site distinct from the ligand binding site and this binding induces desensitization of the receptor (Albuquerque et al., 1997
; Bakry et al., 1988
). Increased binding densities of
4ß2-specific nAChR ligand in the cortex in the present study at 0.001 LD50 suggest that if the concentration of sarin reaches a high enough level in the cell, it may cause allosteric changes in the receptor conformation, exposing higher ligand binding sites. Whether or not a high ligand binding state of the receptor continues to be active long enough or becomes desensitized remains to be discovered. Alternatively, the number and or function of the receptor increased by sarin can be mediated by second messengers such as c-AMP-dependent or independent mechanisms (Gurantz et al., 1993
; Margiotta, 1987
) or by changes in intracellular Ca2+ concentration. An interesting possibility could also involve upregulation of the nAChR as a consequence of hypothermia induced by sarin (Clement, 1991
) because low temperature causes upregulation in surface expression of nAChR (Cooper et al., 1999
).
Muscarinic acetylcholine receptors in the CNS are comprised of 5 distinct classes of receptors (m1-m5). These receptors have distinct structural and pharmacological features and show differential cellular localization (Levey et al., 1991). These receptors are coupled to different G-proteins to transduce cellular signaling from the cell surface. m2-mAChR is coupled to Gi protein, leading to inhibition of adenylate cyclase (Hulme et al., 1990
). Our laboratory has previously shown that chlorpyrifos oxon binds to m2-mAChR in vitro and inhibits cAMP accumulation (Huff and Abou-Donia, 1995
; Huff et al., 1994
). Studies by Ward et al. (1993) and Silveira et al. (1990) also have shown that organophosphate compounds selectively regulate m2-mAChR ligand binding. The data in the current study showing increased m2-mAChR-specific ligand binding in cortex following sarin treatment suggest in vivo regulation. Similarly, studies by Chaudhuri et al. (1993) and Liu and Pope (1996) reported an increased m2-mAChR ligand binding in response to chlorpyrifos treatment. It has been previously shown that presynaptic m2-mAChR could regulate acetylcholine release via a feedback inhibitory mechanism (Marchi et al., 1990
; Raiteri et al., 1984
) and in rat striatal cells, paraoxon inhibited forskolin induced cAMP synthesis, an effect which was blocked by atropine (Jett et al., 1991
). These results suggest that selective effects of sarin on m2-mAChR may have modulatory effects on other processes, such as acetylcholine release, second messenger system, etc. that could influence the toxicity of sarin.
In summary, our results suggest that BChE activity in plasma remains inhibited up to 1520 h following a single LD50 dose of sarin, whereas the brain regional AChE shows differential response to sarin treatment. ChAT activity was induced in the cortex followed by midbrain and brainstem. The increased ChAT activation may cause persistent long-term sarin toxicity even after AChE activity has recovered. Furthermore, our results indicate that sarin caused increased nAChR and m2-mAChR binding in the cortex after 6, 15, and 20 h of single-dose LD50 treatment. Finally, our data clearly suggest that sarin-induced neurotoxicity has multiple mechanisms. The eventual manifestation of sarin toxicity is primarily a dysregulation of the cholinergic system.
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ACKNOWLEDGMENTS |
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NOTES |
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