Inhibitory Effects of Paraquat and Its Related Compoundsonthe Acetylcholinesterase Activities of Humanerythrocytes and Electric

Agric. BioL Chem., 51 (8), 2131-2138, 1987 2131

Inhibitory Effects of Paraquat and Its Related Compoundson the Acetylcholinesterase Activities of HumanErythrocytes and Electric Eel {Electrophorus electricus) Yasuo Seto and Toshiaki Shinohara 2nd Chemistry Section, National Research Institute of Police Science, Sanban-cho, Chiyoda-ku, Tokyo 102, Japan Received March ll, 1987

Paraquat inhibited the acetylcholinesterase activity of humanerythrocytes and electric organs of Electrophorus electricus. The inhibition of acetylcholinesterase activity was reversible, as shown from the following two experimental results: [I] The degree of inhibition was not affected by changing the preincubation time of the enzymeand paraquat before the addition of the substrate. [II] The enzyme, preincubated with paraquat and subsequently freed from inhibitor by gel filtration on Sephadex G-2'5, showed the same activity as the untreated enzyme. Paraquat gave effective protection against the inhibition by an irreversible anionic site inhibitor, dibenamine, but not by irreversible esteratic site inhibitors, dichlorvos and methanesulfonyl chloride. These results indicate that paraquat functions as a reversible inhibitor for the anionic site. The inhibitory powers and Hill coefficients of paraquat and diquat were compared with the other quaternary ammonium compounds.Although secondary to edrophonium, paraquat strongly inhibited acetylcholinesterases of human erythrocytes and electric eel, and showed higher inhibition selectivity for both acetylcholinesterases than for human plasma butyrylcholinesterase. The Hill coefficients concerning the interaction of paraquat with acetylcholinesterases of humanerythrocytes and electric eel were given as 0.83 and 0.73, respectively. This indicates negative cooperativity between these enzymes and paraquat, which is similar to the case with d-tubocurarine. On the other hand, diquat showed weak inhibitory power and low inhibition selectivity, and its Hill coefficients were almost 1.0, indicating a competitive inhibition mode.

Paraquat (1, 1 /-dimethyl-4,4/-bipyridinium hydrate metabolism.5) dichloride), which is widely used as nonselec- Apart from the toxic mechanismof para- tive contact herbicide, has recently raised the quat through its behavior as an oxido-redox incidence of fatal toxicity in man and animal. compound, it seemed appropriate to inves- Manystudies of paraquat toxicity have been tigate the pharmacological effect of paraquat carried out with respect to functional damage in an in vitro experimental system, particularly to the lung, i.e., pulmonary edema leading to since the pharmacological effect of paraquat interstitial fibrosis.1* The biochemical mech- on the central nervous system was yet un- anism for the toxicity can be explained in known. Recently, we reported that paraquat terms of the peroxidation of membranelipids, inhibited acetylcholinesterase (AChE, EC which is mediated through the transfer of a 3.1.1.7) activity of human erythrocytes, but single electron from reduced paraquat to mo- not acylcholinesterase (BuChE, EC 3.1.1.8) lecular oxygen and the subsequent formation of humanplasma in an in vitro experiment.6* of a superoxide anion.2) The other biochemical The former enzymeis generally found in the effects of paraquat on animals have been re- erythrocyte membraneand in neuronal syn- ported to include diminution in lung sur- apses, and classified as a different enzyme from factant,^ inhibition of microsomal mixed- BuChE.7) In the present study, we have clari- function oxidase4) and disturbance in carbo- fied the inhibitory effects of paraquat and its 2132 Y. Seto and T. Shinohara structurally related compoundson the AChEs stopped by adding 70jul of a 12mMeserine solution. For of human erythrocytes and electric eel the blank test, the eserine solution (70/il) was added prior (Electrophorus electricus). to the enzyme reaction. The absorbance of the solution containing the liberated 5-thio-2-nitrobenzoate.was measured at 405 nm. ChEactivity was calculated from a value MATERIALS AND METHODS of the extinction coefficient (a, 13500), and expressed as U/ml. One unit (U) represents the ChE activity hydrolyz- Chemicals. The following chemical reagents were pur- ing 1 /imol of substrate per min. For the assay of electric chased from WakoPure Chemical Industries (Osaka, eel AChE, 30^1 of 10% (v/v) Triton X-100 was added to Japan): acetylthiocholine (ATCh) iodide, butyryl- the reaction mixtureto stabilize the enzymeactivity. thiocholine iodide, 5,5/-dithio-bis(2-nitrobenzoic acid) In the inhibition assay for ChEactivity, an inhibitor was (DTNB), eserine (physostigmine) sulfate, dichlorvos added prior to the preincubation (10min) of the enzyme (dimethyl 2,2-dichlorovinyl phosphate), tetramethyl- reaction. The enzymesamples used were humaneryth- ammonium chloride and trimethylphenylammonium rocyte ghosts (70/ig protein/ml, 40 ~48mU/ml), electric chloride. Dibenamine (A^TV-dibenzyl-^-chlorethyl- eel AChE (400ng protein/ml, 140~180 mU/ml) and hu- amine) hydrochloride and methanesulfonyl chloride man plasma (diluted 50-fold, 1.02~ 1.25mg protein/ml, were purchased from Nakarai Chemicals (Kyoto, 40~63 mU/ml). The inhibitors used were dissolved in Japan). Edrophonium (ethyl[m-hydroxyphenyl]dimethyl- distilled water, except for dichlorvos, dibenamine and ammonium) chloride, electric eel AChE(Type IV-S) and methanesulfonyl chloride, the latter three compounds bovine serum albumin (fraction V) were purchased from being dissolved in absolute ethanol. The final ethanol Sigma Chemical Co. (St. Louis, U.S.A.). d-Tubocura- concentration was minimized in the enzyme reaction mix- rine chloride was obtained from Tokyo Chemical Co. ture within 3% (v/v). ChE activity without an inhibitor (Tokyo, Japan), Sephadex G-25 (fine) was obtained was assayed in the presence of an equal volume ofethanol from Pharmacia Fine Chemicals (Uppsala, Sweden). for a control test. The rate ofthiocholine formation in this Paraquat dichloride and diquat (l,r-ethylene-2,2'-bi- assay proceeded linearly up to 20 min, whether a reversible pyridinium) dibromide were supplied from ICI Japan inhibitor waspresent in the reaction mixture or not. The Ltd. (Tokyo). The other chemicals used were of analyti- level of ChE inhibition, the ratio of the ChE activities in cal reagent grade. the presence or absence of an inhibitor, did not vary during the 15 min reaction time. The inhibition level, given Preparation and solubilization of human erythrocyte as the I50 value, was expressed as the concentration of ghosts. Erythrocyte ghosts were prepared according inhibitor necessary to cause 50%inhibition. to the procedure of Dodge et al.8) using outdated human Hill coefficients and ] erythrocytes in transfusion, and used for a smaple of using the Hill equation1 erythrocyte AChE. Membrane-bound AChE was solubilized by combining one volume of the packed erythro- log[VojVi) - 1] =nH log[I] +constant cyte ghosts with three volumes of 0.167m Tris-HCl where Voand Vi are the activity in the absence and in the buffer (pH 6.8) containing 0.667% (v/v) Triton X-100, presence of an inhibitor, [I] is the inhibitor concentration, and then stirring at 2°C for 1 hr. The solubilized solu- and nH is the Hill coefficient. By plotting \og[(Vo/Vi)- 1] tion was centrifuged (100,000xg) at 2°C for 1hr, and against log[I] and by data analysis with the least-square the supernatant thus obtained was used for the experiment. linear regression, each nH value was obtained from the slope of the fitted linear line. Also, the I50 value was Cholinesterase assay. Cholinesterase (ChE) activity was obtained from the inhibitor concentration corresponding measured by the method of Ellman et al.9) with some to a value of0 on the \og[(Vo/Vi)-1] axis, as shown in modification, as previously described.10' For AChE ac- Fig.2. tivity, human erythrocyte ghosts and electric eel AChE were used as the enzyme sources. For BuChE activity, Inactivation of acetylcholinesterase. The solubilized so- humanplasma isolated from outdated humanblood was lution of humanerythrocyte ghosts (final concentration of used. Onehundred iA of a diluted sample was added to 0.16mg protein/ml, 0.29 U/mg of protein) was incubated 3ml of a 0.25mM DTNB solution containing 20mM at 25°C with or without an inhibitor in a buffer solution sodium phosphate (pH 7.7), and the mixture was prein- (pH 7.7) containing 20him sodium phosphate and 0.1% cubated for lOmin at 25°C while being shaken. The (v/v) Triton X-100 (buffer A). For electric eel AChE, the enzymereaction was started by adding a substrate to the enzyme solution (final concentration of 2 ^g protein/ml) preincubated solution, 10/il of 156mMATCh solution for was incubated in buffer A. The inhibitors used were AChE and 40/zl of 156mM butyrylthiocholine iodide paraquat, dichlorvos, methanesulfonyl chloride and di- solution for BuChEbeing added as the substrate reagent. benamine. The inhibitor was removed from the inhibitor- Such a mixed assay solution was further incubated at 25°C conjugated AChEby gel filtration on a Sephadex G-25 for 15minwhile being shaken. The enzymereaction was column equilibrated with buffer A at 2°C. An aliquot Acetylcholinesterase Inhibition by Paraquat 2133 (100jul) of the incubation mixture was applied on the increased during the preincubation time fror Sephadex G-25 column (1 x 22cm), and then eluted with 0 to lOOmin, indicating an 'irreversible' in buffer A at a flow-rate of 4~5ml/hr. The void volume hibition. On the contrary, the degree of ACh] fraction containing the inhibitor-conjugated enzyme was collected. To the fraction (3.18ml) preincubated at 25°C inhibition by paraquat and diquat did nc for lOmin, IOOjuI of 10mMDTNBsolution and 10/4 of change over a period of lOOmin. 156mMATChsolution was added, and the mixed solution As shown in Table I, erythrocyte AChl was further incubated for 15min. The enzyme reaction preincubated with dichlorvos and sub was stopped by adding 70fi\ of 12mMeserine solution, and the AChEactivity was measured as already described. sequently separated from the excess inhibito In the experiment for the 'protective effect' ofparaquat, lost its activity to a great extent (about 67%c the enzyme solution was incubated with an irreversible the AChE activity in the absence of an in inhibitor and paraquat. The detailed incubation con- hibitor), while erythrocyte AChE preincubate< ditions are described in the legend to Table II. with paraquat and subsequently separate< Protein assay. The protein contents of human plasma from excess inhibitor retained the same ACh] and erythrocyte ghosts were determined by a biuret reacr tion12) in the presence of 1.0% (w/v) sodium deoxy- Table I. Reversibility of Acetylcholinesterase cholate in order to remove any turbidity in the sample Inhibition by Paraquat solution. The microassay of the protein content in the H u m an eryth rocyte E lectric eel In hibitor solubilized solution of erythrocyte ghosts was carried A C h E A C h E out by the method of Lowry et al.13) with the correction of an error caused by contamination by the detergent N o ne 100 100 (Triton X-100). Bovine serum albumin was used as a P ara quat 103.0 98.5 standard protein. D ichlo rvos 33.0 17.8

RESULTS The time of incubation with an inhibitor was 30min, and the final inhibitor concentration in the incubation mixture was 359^m for paraquat and 113/iM for dich- Reversibility of A ChE inhibition lorvos. Detailed experimental conditions are described in As shown in Fig. 1, the degree of inhi- Materials and Methods. Each value represents the bition against the AChE activity (human percentage of AChEactivity compared with the activity stroma, A; electric eel, B) by dichlorvos was without inhibitors.

ioot ' h 1001 . n

r> \ V A il^D-0\ D D D D B D

> [ln° ^V° m E \

à"åo (;% ^50L.à"à" ^^^ V^°; V t g- 50- \

O à" à" . å --å .å å å < å °0 5Q 100 °0 50 100 Incubation time Cmin3 Fig. 1. Change of Acetylcholinesterase Activity with Preincubation Time in the Presence of Paraquat and Dichlorvos. Enzyme solutions (100^1 containing 7 /ig of human stroma protein, 15 mU(A); 40 ng of protein of electric eel AChE, 135mU(B)) were mixed with 3ml of 0.25mMDTNBsolution containing 20mMsodium phosphate (pH 7.7) in the presence ofparaquat (# A, 30.9/im; B, 3.08/zm), diquat (å¡ A, 1.25mM; B, 125/^m) and dichlorvos (å A, 0.721 /zm; B, 1.43fiM). After incubation (0~ 100min) at 25°C with an inhibitor, the AChE activities were measured. The residual activity is expressed as a percentage comparedwith the activity without an inhibitor. 2134 Y. Seto and T. Shinohara

Table II. Protective Effect of Paraquat against the Irreversible Inhibition OF ACETYLCHOLINESTERASE

H um an erythrocyte E lectric eel A C h E A C hE

C on trol 100 100 E xp . 1 D ibenam ine 18.5 18.3 Dibenamine + paraquat 82.3 86.7

C on trol 100 100 E xp . 2 D ichlorv os 33.0 17.7 Dichlorvos + paraquat 24.8 15.4

C on tro l 100 100 E xp . 3 M S C * 44.7 15.1 M SC +par aqu at 44.7 0.0

Incubation conditions were as follows: Exp. 1, each enzymesolution was incubated for 170min at 25°C with dibenamine (495/zm) alone or with dibenamine (495 ^m) and paraquat (5.29him); Exp. 2, for 30min with dichlorvos (113/zm) alone or with dichlorvos (113/^m) and paraquat (970^m); Exp. 3, for 30min with methanesulfonyl chloride (6.14mM) alone or with methanesulfonyl chloride (6.14mM) and paraquat (44.5mM). An aliquot of the incubation mixture was applied to gel filtration. The experimental conditions are described in Materials and Methods. Each value represents the percentage of AChEactivity compared with the activity in the absence of the inhibitors. a Methanesulfonyl chloride. activity as in the control experiment without tubocurarine and edrophonium were linear an inhibitor. A similar result was obtained in within a concentration range from about - 1 the experiment with electric eel AChE(Table to about 1 on the \og[(Vo/Vi)-1] axis (cor- I). responding to about 10~90% inhibition of enzyme activity). Table III illustrates the I50 Binding site ofparaquat in the AChEmolecule values of the quaternary ammoniumcom- As shown in Table II, dibenamine, di- pounds for humanerythrocyte AChE,electric chlorvos and methanesulfonyl chloride pro- eel AChE and human plasma BuChE ac- duced inactivation of both human erythro- tivities. These compounds mostly showed cyte and electric eel AChEs. However, the stronger inhibitory effects on AChEs of hu- addition of paraquat to the preincubation manerythrocyte and electric eel compared mixture of AChE and dibenamine alleviated with human plasma BuChE. The ratio of I50 the inactivation by dibenamine. On the for human plasma BuChE and that for hu- contrary, the addition of paraquat to the man erythrocyte AChEmay explain the in- preincubation mixture of AChEand dichlor- hibition selectivity of AChE to BuChE. vos (or methanesulfonyl chloride) did not Edrophonium, known as a muscle stimulant, retard the extent of such inactivation. More- had the strongest inhibitory power and the over, the levels of inactivation of dichlorvos highest inhibition selectivity on AChE activity and methanesulfonyl chloride were in- among the quaternary ammonium com- creased by adding paraquat. pounds. Paraquat possessed strong inhibitory power, the same level as that of d-tubo- Comparison of150 values with the other quater- curarine, known as a skeletal muscle relax- nary ammoniumcompounds ant, and had inhibition selectivity as high as As shown in Fig. 2, Hill plots of the in- trimethylphenylammonium. On the other hibition against human erythrocyte (A) and hand, diquat showed weak inhibition selec- electric eel (B) AChE's by paraquat, d- tivity, and its inhibition power was between Acetylcholinesterase Inhibition by Paraquat 2135

Table III. Comparison between the Inhibitory Powers of Quaternary AmmoniumCompoundson Cholinesterases C h E

In h ib ito r H u m a n H u m a n E lectric DR a t.i o ( A) (B ) ery th r o c yte p la sm a e el A C h E (A ) B u C h E (B ) A C h E

P a r aq u a t 2 9 7,9 0 0 6. 1 2 7 0 D iq u a t 1, 50 0 9,8 0 0 5 8 0 6.6 d-T u b o cu ra rin e 6 3 35 0 5 6 5.5 E d ro p h o n iu m 0. 87 1, 30 0 0.3 7 1, 5 0 0 T rim eth y lp h e n y la m m o n iu m 15 0 2 9, 00 0 1 10 19 0 T etra m eth y la m m o n iu m 1 1, 0 0 0 8 7, 00 0 4, 50 0 8. 1

Each value represents I50 (,um).

, ! , , Table IV. Comparison between Hill Coefficients of Quaternary AmmoniumCompounds for acetylcholinesterases A C h E sou rce

In hi b it or TT C l .H u ma n E l ec tr ic eryth rocyte eel \-2 -1 0 1 2 3

S ______P araqu at 0 .83 0 .73 D iqu at 0 .95 1.0 6 fl f-T ubo cu rar in e 0. 69 0 . 78 E d rop hon ium 0 .90 0.96 Tr im et hy lp he ny la mm on iu m 0. 9 4 0 . 96 T etram eth ylam m on ium 1.04 0 .9 7

-2 -1 0 1 2 3 log[Inhibitor] CjjMD Comparison of Hill coefficients with the other quaternary compounds Fig. 2. Hill Plot of the Acetylcholinesterase Interaction Hill coefficients of these quaternary am- with Quaternary AmmoniumCompounds. The enzyme solution (100^1 containing 7/zg of human moniumcompounds just mentioned are sum- stroma protein, 4.0~4.8mU (A); 40ng of protein of marized in Table IV. The values of d- electric eel AChE, 14- 18mU (B)) was mixed with 3ml of tubocurarine were given as 0.69 and 0.78 for 0.25mM DTNBsolution containing 20mMsodium phos- human erythrocyte and electric eel AChEs, phate (pH 7.7) in the presence of an inhibitor. After 10 min respectively. Paraquat also showed small val- of incubation, 10/i of 156mMATChsolution was added ues, less than 1.0, for both AChEs, which to the mixture, which was further incubated for 15 min at indicates negative cooperativity, the same as 25°C. The reaction was stopped by adding 70/d of 12mM eserine solution. The residual AChE activity (Vi) was that of J-tubocurarine. On the other hand, compared to that without an inhibitor ( Vo). The inhibitors the Hill coefficients of the other compounds, used were paraquat (#), J-tubocurarine (A) and edro- diquat, trimethylphenylammonium, tetra- phonium (A). The straight lines were fitted to these data methylammonium and edrophonium, were by the least-squares linear regression. all close to 1.0 for both AChEs. that of trimethylphenylammonium and tetra- DISCUSSION methylammonium. Most organophosphorus and carbamate 2136 Y. Seto and T. Shinohara pesticides are known to inhibit ChEactivity by activity with the other quaternary ammonium covalent binding to the active center of the compounds. Edrophonium showed the highest ChE molecule, resulting in irreversible inhibi- inhibitory power and the highest inhibition tion.14) On the other hand, from our two selectivity on AChEactivity. This strong in- experimental results (Fig. 1 and Table I) we hibition probably derives from its molecular can conclude that AChE inhibition by the structure: the quaternary ammoniumportion herbicides, paraquat and diquat, is reversible. has a binding affinity to the anionic site, and Because the ChE molecule is known to the hydroxyl group sterically positioned to the possess two sites in its active center,15) ChE esteratic site forms a hydrogen bond, perhaps inhibitors can be divided into two groups: an with the imidazole group of the esteratic anionic site inhibitor and an esteratic site site.22) Although secondary to edrophonium, inhibitor, although some inhibitors have bind- paraquat showed stronger inhibitory power ing affinity for both sites. Dibenamine is con- and higher inhibition selectivity on AChE verted to ethyleniminium ions possessing activity. Inhibition selectivity refers to the dif- quaternary ammoniumion structures, and ference between the binding affinity of the forms reversible addition complexes with the inhibitor to the anionic site of each ChE anionic site, and binds covalently to AChE at molecule. It is concluded that paraquat bound or near the anionic site (alkylation)16); meth- to an anionic site of AChE strongly and anesulfonyl chloride17) and dichlorvos14) are selectively, compared to BuChE. covalently bound to the hydroxyl group of It has been reported that some divalent serine in the esteratic site. Table II shows that quaternary ammoniumcompounds can bind paraquat bound to the anionic site and pro- to two sites of AChE, not only to the anionic tected sterically against the attack of an irre- site of the active center, but also to another versible inhibitor on the anionic site, but did anionic site.23) The latter site may be an allo- not protect against the attack on the esteratic steric (regulatory) site, which causes a con- site. This results suggests that paraquat com- formational change to the AChEmolecule, petitively bound to the anionic site, which is resulting in an alteration of the AChEactive in agreement with the observation of Belleau site.24) d-Tubocurarine, a typical pachycurare, and Tani,18) who demonstrated that tetra- gave a value less than 1.0 for the Hill coef- methylammonium iodide, a competitive in- ficients, which is compatible with the reports hibitor, retarded irreversible inactivation by of Moss et al.25) and Zorko et al.26) As the 7V,A^-dimethyl-2-chloro-2-phenethylamine. Hill coefficients for paraquat were less than The level of the inhibitory effect of the ir- 1.0, it is possible to assume that paraquat can reversible esteratic inhibitors was increased bind not only to the anionic site of the active with the addition of paraquat. This is com- center but also to another site, which sug- patible with other reports,19'20* which dem- gests negative cooperativity. On the other onstrate that masking of the anionic site by hand, diquat showed about 1 for the Hill co- some quaternary ammonium ion had a efficients, indicating that it bound only to an marked accelerating effect on the rate of anionic site of the active center in both en- sulfonylation19) and carbamylation20) of the zymes. esteratic site. Toxicologically, it has been reported that Somekinds of quaternary ammoniumcom- the peak concentration of paraquat in blood poundsare well knownas reversible inhibitors was 22/ig/ml for rat, 5jUg/ml for guinea-pig possessing an affinity for the anionic site of the and 70/^g/ml for cat.27) Because the I50 value active center.21* Because paraquat and diquat of paraquat for humanerythrocyte AChEwas are regarded as quaternary ammoniumcom- 29/llm (lA fig/ml) in a clinical case ofparaquat pounds, it may be significant to compare the poisoning, it is uncertain whether the eryth- inhibitory effects of these herbicides on ChE rocyte AChE is inhibited in vivo. Also, the Acetylcholinesterase Inhibition by Paraquat 2137

physiological function of erythrocyte AChE M. S. Rose, H. C. Crabtree, K. Fletcher and I. has not been clearly elucidated.28) From the Wyatt, Biochem. J., 138, 437 (1974). T. Shinohara and Y. Seto, Agric. Biol. Chem., 50, 255 results of this experiment, it remains uncertain (1986). whether the toxicity of paraquat is related to International Union of Biochemistry, "Report of the the inhibition of erythrocyte AChEactivity or Commission on Enzyme 1961," Pergamon Press, not. Oxford, 1964, p. 104. It seems plausible that paraquat can in- J. T. Dodge, C. Mitchell and D. J. Hanahan, Arch. hibit the nervous AChE influentially in ad- Biochem. Biophys., 100, 1 19 (1963). G. L. Ellman, K. D. Courtney, V. Andres, Jr. and R. dition to the inhibitory effect on human eryth- B. Featherstone, Biochem. Pharmacol., 7, 88 (1961). rocyte and electric eel AChEs. However, it T. Shinohara and Y. Seto, Repts. Natl. Res. Inst. may be difficult to infer that paraquat can Police Sci., 38, 178 (1985). R. B. Loft field and E. A. Eigner, Science, 164, 305 enter the brain through the blood-brain barrier (1969). because of its cationic and hydrophilic nature. A. G. Gornall, C. J. Bardaeill and M. M. David, J. Litchfield et al. demonstrated that in rats, both Biol. Chem., Ill, 751 (1949). paraquat and diquat were rapidly distributed 0. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. throughout most tissues except for the brain Randall, J. Biol. Chem., 193, 265 (1951). and spinal cord.29) However, ifparaquat partly W. N. Aldridge, "Enzyme Inhibitors as Drugs," ed. enters the cholinergic junctions in the pe- by M. Sandier, MacMillan Press, London, 1980, pp. 115-125. riphery, it maydisturb normal nervous trans- 1. B. Wilson and F. Bergmann, /. Biol. Chem., 185, mission at the synapses. Accordingly, the tox- 479 (1950). icity of paraquat and diquat may be as- F. Beddoe and H. J. Smith, Nature, 216, 706 (1967). sociated with the inhibition of AChE. H. C. Froede and I. B. Wilson, "The Enzymes," 3rd Recently, 1-methyl-4-phenyl-1,2,3,6-tetra- Ed., ed. byP. H. Boyer, Vol. 5, Academic Press, New hydropyridine (MPTP) has been found to in- York, 1971, pp. 87-114. B. Belleau and H. Tani, Mol. Pharmacol., 2, 411 duce a parkinsonian-like syndrome in hu- (1966). mans and some animals,30) and its neuronal R. Kitz and I. B. Wilson, /. Biol. Chem., 238, 745 toxicity is explained as being derived from 1- (1963). methyl-4-phenylpyridinium (MPP+) that is H. P. Metzger and I. B. Wilson, J. Biol. Chem., 238, 3432 (1963). converted from MPTP by the action of monoamineoxidase.31) As paraquat is so I. B. Wilson, "The Enzymes," 2nd Ed., ed. by P. D. Boyer, H. Lardy and K. Myrback, Vol. 4, Academic structurally similar to MPP+, it is thought to Press, New York, 1960, pp. 501 -520. have a toxicity similar to MPP+.32'33) Consid- I. B. Wilson and C. Quan, Arch. Biochem. Biophys., ering the two observations of the neuronal 73, 131 (1958). J-P. Changeux, Mol. Pharmacol., 2, 369 (1966). toxicity of MPP+and the similarity of the B. D. Roufogalis and E. E. Quist, Mol. Pharmacol., chemical structure, it will be of much phar- 8, 41 (1972). macobiochemical interest to investigate the D. R. Moss, D. E. Moss and D. Fahrney, Biochim. effect of paraquat on the nervous system such Biophys. Acta, 350, 95 (1974). as the cholinergic and dopaminergic neurons. M. Zorko and M. R. Pavlic, Biochem. Pharmacol., 35, 2287 (1986). D. M. Conning, K. Fletcher and A. A. B. Swan, Br. REFERENCES Med. Bull, 25, 245 (1969). A. Silver, "The Biology of Cholinesterases," North- 1) A. Pasi, "The Toxicology of Paraquat, Diquat and Holland Publishing Co., Amsterdam, 1974, pp. Morfamquat," Hans Huber Publishers, Switzerland, 358-364. 1978. M. H. Litchfield, J. W. Daniel and S. Longshaw, 2) J. S. Bus, S. D. Aust and J. E. Gibson, Biochem. Toxicol., 1, 155 (1973). Biophys. Res. Commun., 58, 749 (1974). J. W. Langston, P. Ballard, J. W. Tetrud and I. Irwin, 3) B. W. Manktelow, Br. J. Exp. PathoL, 48, 366 (1967). Science, 219, 979 (1983). 4) R. I. Krieger,P. W. Lee,A. BlackandT. R. Fukuto, J. A. Javitch, R. J. D'Amato, S. M. Strittmatter and Bull. Environ. Contam. ToxicoL, 9, 1 (1973). S. H. Snyder, Proc. Natl. Acad. Sci. U.S.A., 82, 2173 2138 Y. Seto and T. Shinohara

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