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The effect of steric factors on the interaction of some phenylphosphonates with acetylcholinesterase and neuropathy target esterase of hen brain
PESTICIDE
BIOCHEMISTRY
AND
PHYSIOLOGY
25, 133- 142 (1986)
The Effect of Steric Factors on the Interaction of Some Phenylphosphonates with Acetylcholinesterase and Neuropathy Target Esterase of Hen Brain M. K. JOHNSON,]
D. J. READ,
AND H. YOSHIKAWA*
Molecular Toxicology Section, Toxicology Unit MRC Laboratories. Woodmansterne Road. Carshulton. Surrey, SM5 4EF, England; and *Deportment of Environmental Chemistry. Furldry ofEngineering, Kyushu Kyoritstt University, Kitukyushrt 807. Japan
Received January 14. 1985; accepted April 12, 1985 At 37°C and pH 8.0 the resolved isomers of S-(4-chlorobenzyl or 2.4-dichlorobenzyl) ethyl phenylphosphonothiolate are moderate inhibitors of AChE of hen brain (k, = 300-1400 M-‘min-‘) and weak inhibitors of neuropathy target esterase (NTE) (k, = 40-240 M-‘min-I). The two (R),( +) isomers are more active against NTE than are the (S),( - ) while the opposite is true for AChE. NTE inhibited by either (R),( + ) isomer underwent slow spontaneous reactivation (about 30% in 18 hr) but (S),( - )-inhibited NTE did not reactivate. No spontaneous reactivation was detected for AChE inhibited by either steric form. AChE inhibited by (R)p( + ) isomers could be reactivated by N-methylpyridinium-2-aldoxime methanesulfonate and slow aging was detected (23% in 18 hr). NTE inhibited by (R)p( + ) isomers could be reactivated by o-isonitrosoacetophenone and no significant aging occurred in 18 hr at 37°C. Neither of the enzymes could be reactivated 60 min after commencement of inhibition with saturated solutions of the (S)p( -) isomers. NTE inhibited by racemic EPNO in a I-min incubation formed a mixture of rapidly aging (tic = I min) and apparently non-aging inhibited NTE. Since EPNO should form the same ethyl phenylphosphonylated enzymes as the isomers under study we conclude that the oxime-resistance of inhibited NTE after inhibition by these (S)p( - )-isomers is due to rapid formation of aged enzyme during the (necessarily) long inhibition period. Substantial inhibition of NTE in viva should be possible in hens protected against cholinergic effects of a dose of either (R),( + ) isomer. The toxicological consequence of such inhibition (? neuropathy or protection) should be investigated. %’ 1986 Academic Press, Inc.
INTRODUCTION
Two major toxic effects of OP* esters are known. These are the acute toxicity initiated by covalent organophosphylation of i To whom correspondence should be addressed. * Abbreviations used: OP, organophosphorus; AChE, acetylcholinesterase; NTE, neuropathy target esterase, formerly called neurotoxic esterase; INAP, w-isonitrosoacetophenone; P2S, N-methylpyridinium 2-aldoxime methanesulphonate; MCB, ethyl S-[4chlorobenzyl] phenylphosphonothiolate; DCB. ethyl S-[2,4-dichlorobenzyl] phenylphosphonothiolate; EPNO (the oxon of the pesticide EPN) ethyl 4-nitrophenyl phenylphosphonate.
the active centre of AChE and delayed neuropathy initiated by a similar reaction on the active site of NTE (l-4). For delayed neuropathy a second essential step in the initiation process is aging of the inhibited enzyme (Reaction (4) in Scheme 1) (5, 6). The structural features in OP esters associated with inhibitory power against AChE and NTE are different (7-9) so that some anticholinesterase compounds are known which are neuropathic below LD,, while others are not neuropathic in animals even at doses far above unprotected LD,, given in conjunction with prophylaxis and therapy against acute toxicity.
133 0048-3575/86 $3.00 Copyright 0 1986 by Academic Press. Inc All rights of reproduction in any form reserved
134
JOHNSON,
O
E OH+.-!’
SCHEME 1. Steps Michaelis complex; (4) aging.
OR
(1)
[
READ,
AND
0 II/
EoH---x-P,~~
in the interaction (2) phosphorylation
OR
YOSHIKAWA
1
*
(2)
0
--‘Eo-P;
OR I
of an esterase with an organophosphorus of enzyme; (3) reactivation (spontaneous
Many organophosphonates of interest as pesticides or potential chemical warfare agents contain a chiral centre and in some cases the resolved isomers have markedly different toxicities. Thus the two P( -) isomers of soman have 10-100x the acute toxicity to mice of the P( +) isomers while in vitro the anticholinesterase activities differ by at least 1000x (10). Against NTE in vitro the potency of these four isomers differed over a lo-fold range (11). In vivo diffc :nces in neuropathic potency have been reported for isomers of the pesticide EPN (12, 13) and the difference has been attributed to the greater anti-NTE potency of the ( -) isomer of EPNO (14). Only one study of aging of inhibited NTE after inhibition by resolved isomers has been reported (I 1). This was with isomers of soman and we were surprised to find that some structures exist which yielded forms of inhibited NTE which aged slowly: for most of the inhibitory compounds (nonchiral phosphates or racemic mixtures of phosphonates) which had been reported previously, aging of inhibited NTE was very fast (15). Since aging of inhibited.NTE is an essential second step in the initiation of delayed neuropathy, further studies of aging of inhibited NTE are of considerable interest to toxicologists as well as to enzymologists. Some years ago Professor W. Dauterman
OR
”!
inhibitor. orforced
(I) Formation of by oximes or KF);
kindly supplied us with small samples of resolved isomers of EPNO. However, some decomposition had occurred during transshipment and we were unable to do more than preliminary measures of I,, values which agreed well with those subsequently published by Ohkawa et al. (14). However, the resolution of ethyl phenylphosphonothiolic acid and formation of S-esters with retention of configuration is now well established and relatively easy to perform. Studies on anti-cholinesterase and insecticidal activity of such resolved chiral esters have been reported (16). Although the thioloester bond is not a favored structural feature for NTE inhibition, the phenylphosphonate moiety is (7). From a wide range of available structures we therefore chose the resolved isomers of S-4-monochlorobenzyl and S-2,Cdichlorobenzyl esters as potential inhibitors for study. On the basis of general chemistry and of proof with one S-methyl phosphorothiolate (17) it is reasonable to predict that these compounds should form exactly the same ethyl phenylphosphonylated esterases in vitro as would be obtained when inhibiting with the oxons of EPN, cyanofenphos or ethyl leptophos . We report here on the inhibition and aging of hen brain AChE and NTE using these isomers and seek to interpret the significance for effects in vivo.
PHENYLPHOSPHONATE MATERIALS
AND
METHODS
Chemicals The resolved and purified [(R)r( +) and (S),( - )] isomers of MCB and DCB were prepared in Japan as described previously (16), shipped by air to the United Kingdom and stored in desiccators at -5°C until required: the dry compounds are very stable under these conditions and optical purity was greater than 98%. Stock solutions in dry acetone were prepared as required. The unresolved oxon of EPN was a gift of Dr. H. Ohkawa, Sumitomo Chemical Company: it was essentially free of 4-nitrophenol. INAP was from Aldrich Chemical Company and melted at 127°C (lit 126128°C); an older sample melted over a wide range and, while it appeared active as a reactivator, it was not used for definitive experiments. P2S was a gift from Chemical Defense Establishment, Porton Down. Buffers. For AChE assay solutions of NaH?PO, and Na,HPO, (each 100 mA4) were mixed to give the desired pH. For most other experiments Tris (50 mM)/ EDTA (0.2 mM) was adjusted to pH 8.0 with HCI (about 12 M) at ambient temperature. To prepare the reactivation medium for NTE 1 vol of INAP (500 mM in acetone) was added to 24 vol Tris base (50 mM)l EDTA (0.2 m&f) at 37°C and the pH was adjusted to 9.0 with HCl: the INAP acts as a buffer in the pH range 8-9 and is probably a more effective reactivator in its ionized form. Inhibition, Aging, Reactivation, and Assay of NTE Detailed protocols are given in Ref. (11) and brief comments are given here. The activity of NTE is normally measured as the difference in phenyl valerate esterase activity between two samples which have been preincubated with a non-neuropathic progressive inhibitor (paraoxon or benzenesulphonyl fluoride) with or without the neuropathic inhibitor, mipafox. The rea-
ISOMERS
AND
NTE
135
sons for the choice of inhibitors used in aging studies were presented in Refs. (6) and (15). We obtained improved dispersion of sedimented particles prior to assay by including Triton X-100 (0.01%) in the final dispersion medium as described recently (11). (a) Study of time course of inhibition. Paired tissue samples, routinely called “P” and “M,” were prepared and used exactly as described (11, 6); the samples are identical except that P contains active NTE while M does not. Inhibitors were added from concentrated stock solutions in acetone and the final solvent concentration was not more than 1% (v/v). (b) Study of spontaneous reactivation and aging. (i) For the situation when the aging period was more than 1 hr the improved procedure of Ref. (11) was used in which inhibited enzyme was separated from unreacted inhibitor by sedimentation and resuspension in fresh buffer before the commencement of the timed aging period. In initial experiments a confusing amount of ongoing inhibition occurred during the 18-hr aging period at 37°C. Thereafter the inhibited particles were washed by one or two cycles of resedimentation and resuspension. (ii) For the cases when aging was rapid and the inhibition period limited to a few minutes the procedure of Ref. (6) was used except that the first dilution step ( x 20) after a 1-min inhibition used ice-cold pH 8.0 Tris buffer to halt the progress of inhibition more effectively during the 30set delay before a further 4.1 x dilution into the reactivation medium at 37°C. The nucleophilic reactivator was INAP and not KF as used previously: inhibited tissue (8 ml in pH 8 buffer) was mixed with 25 ml of a solution of INAP [20 mM in acetone/pH 9 buffer (4:96 v/v)] and incubated at 37°C for 30 min prior to cooling and sedimentation of particles etc. as before (11). The high concentration of acetone was necessary to prevent crystallization of INAP during the cold centrifugation.
JOHNSON,
136
READ,
AND
Inhibition, Aging, and Reactivation of AChE Inhibition and Ellman assay procedures were as fully described before (11). Manipulation of tissue for aging and reactivation was exactly as for NTE except that P and M samples were not needed. The reactivation procedure used P2S (1 mM final at pH 8.0 for 20 min at 37°C).
YOSHIKAWA AChE
NTE
2.0 1.9 1.8 1.7 1.6 1.5 1.4
RESULTS
Inhibition of Enzymes As observed by eye the solubility limit of the inhibitors in buffer at 37°C was about 200 pJ4. Figure 1 shows semi-log plots of enzyme activity vs time of preincubation with inhibitor. These were linear and the first-order rate constants derived from the slopes of the lines were approximately proportional to inhibitor concentration up to the solubility limit. Against NTE the (S)p( -) isomers were so weak that even at 100-200 PM the slopes of the first order plots were very shallow. The second order rate constant for inhibition (k,) was derived from the slope (k’) of each line in Fig. 1 using the relationship: k, =
k’
-2.303
[Cl
where [Zj is the molar concentration of inhibitor. Mean values are given in Table 1 which shows that in each case the compounds were more active against AChE than against NTE. Against AChE the (S),( -) isomers were 2-3 x more active than were the (R)p(+) isomers but against NTE (R)r( +) isomers were more active than (S),( -). It follows that the ratios k NTEIkaAChEdiverged greatly being about 0:4 for (R)r(+) isomers and 0.03-0.09 for W,( - 1. Spontaneous Reactivation of Inhibited NTE and AChE Table 2 shows that there was no evidence for spontaneous reactivation of NTE inhibited by (S),( -) isomers: activity at 18 hr
.p 1.7 .c
1.6
E
1.5
1.9 1.8 1.7
MC8 SIpI
1.6 1.5 1.4
t....
10
Inhibition
Time
20
30
40
(min)
FIG. 1. Semi-lag plots of progressive inhibition of hen brain AChE and NTE by isomers at concentrations shown and at pH 8.0 and 37°C. For AChE residual activity was determined after preincubation of tissue with inhibitor for periods up to 40 min. For NTE paired tissue samples differing only by containing active or inactivated NTE were prepared as described under Materials and Methods. These paired samples were further preincubated with test inhibitor for periods up to 40 min and the residual phenyl valerate esterase activity was measured and the NTE activity calculated by difference. Slopes (k’) of the progress lines were measured and used to derive k, values given in Table 1. Each value of k’ was obtained from a line through 4-6 points: the error in these values of k’ were much smaller than the error of the mean value of k, derived from each family of lines.
PHENYLPHOSPHONATE
ISOMERS
TABLE Second
Order
Rate Constants (k,) Phenylphosphonate
137
AND NTE
1
(M-l min-‘) for Stereoisomers
Inhibition at 37°C.
pH
of AChE 8.0
and
NTE
by
Isomer Enzyme
DCB-(R),( + )
MCB-(R),( + )
DCB-(S),( - )
MCB-(S),( - 1
AChE NTE Approximate ratio of rate constants
560 (3) 240 (3)
300 (4) 120 (3)
1400 (4) 40 (3)
I100 (4) 95 (2)
0.4
0.4
0.03
0.09
k NTE//( AChE a a
Nofe. Mean values of k, were derived from the slopes of lines in Fig 1. Individual values differed from mean by less than 10% except for MCB-(R),(+) against NTE where the range from mean was 16%. The number of determinations is given in parentheses:
was less than that at zero time even when allowance was made for some control losses due to instability. We presume that sufficient traces of lipophilic inhibitor survived the initial sedimentation and washing of inhibited particles to cause further inhibition. For this reason it is impossible to say that no spontaneous reactivation occurred but the results are in clear contrast to those with (R)r( +) isomers where activity increased during 18 hr in spite of the control losses and the possibility of continued inhibition at concentrations similar to those of the (S),( -) isomers. The calculated percentage values for spontaneous reactivation are very crude since they depend on a ratio derived from comparatively small differences between large numbers. However, the effect is sufficiently reproducible to be considered real. For AChE no spontaneous reactivation was detected after inhibition by either isomer. The initial experiments were done in conjunction with aging studies and inhibition was at high concentrations for short times. Latterly we extended inhibition time up to 6 hr to use lower concentration of inhibitor: even this procedure followed by sedimentation of the particles and two resuspension-washes with cold buffer did not entirely eliminate the problem of ongoing inhibition during the 18-hr incubation so that the failure to detect reactivation only shows that the rate. if any, is very low.
Znduced Reactivation and Aging of Inhibited NTE and AChE Most previous studies of aging of inhibited NTE have used KF as reactivator since there is unlikely to be steric hindrance to access of this reactivator to the active site and also the agent is both cheap and easily removed by sedimentation of the particlebound enzyme. Preliminary experiments on reactivation by KF of either NTE or AChE of hen brain after inhibition by the phenylphosphonothiolates showed only massive increases in inhibition. This we attribute to the conversion of residual amounts of the comparatively poor inhibitors to analogous fluoridates which are likely to be much more potent inhibitors: a similar problem has been found previously with transformation of paraoxon and some other nitrophenyl esters in contact with KF (15). We therefore used the well-known P2S instead of KF as AChE reactivator (see Materials and Methods). Clothier (18) found that a saturated solution of INAP, (about 5 mM) was a moderately effective reactivator of diethylphosphinyl-NTE. We have now found that higher concentrations of INAP remain in solution throughout the reactivation and cold centrifugation steps if the medium is made up to pH 9.0 and contains acetone (3% v/v) as described under Materials and Methods section. Incubation for 30 min at 37°C in this reactivation medium
JOHNSON,
138
READ, AND YOSHIKAWA TABLE 2
Spontaneous
Reactivation
of Inhibited
NTE
and AChE
Activity at zero time Inhibition cone (PM)/ time (min)
Isomer NTE DCB-(R),( + 1
(a) Control uninhibited (%I
(b) Inhibited (%o)
during
18 hr at 3X,
pH 8.0
Activity at 18 hr (cl Control (%I
Cd) Inhibited (So)
Spontaneous reactivation dlc
-
bla
%
100 - b
250/60b 250/60b 100/180 601270
100 100 100 100
25 32 14 49
80 74 84 83
34 36 24 53
24 25 17 29
MCB-(R),( + 1
300/60b 100/180 801270
100 100 100
35 38 39
74 84 83
42 55 56
34 44 47
DCB-(S),( - )
800/6Ob 200/180 100/360
100 100 100
56 55 56
80 84 89
22 35 44
-64 -29 -15
MCB-(S),( - 1
800/60b 200/180 100/360
loo loo 100
76 60 56
80 84 89
31 43 46
-154 -23 - 10
+1
500125’ 500125 601270 30/200 201360
100 100 100 100 100
9 9 14 40 41
71 87” 92 96 91
2 70 7 18 23
-7 -la -35 -7 -27
+1
5OOl25’ 500125 801270 50/200 251360
100 loo 100 loo loo
14 11 11 23 34
71 87” 92 96 91
4 15” 12 23 31
-10
- 1
300/2Sb 15/200 201360
100 100 100
36 73 47
69 96 91
3 31 19
-50 -150 -49
MCB-(S),( - )
300125’ 300125 20/200 151350
100 100 100 100
20 21 48 48
69 87“ 96 91
4 13” 34 35
AChE DCWV,(
MCWR),(
DCB-W,(
7” 2 1 0
-18 - 8” -24 -18
Note. Procedure was as described under Materials and Methods: all inhibited and “solvent-inhibited” control particles were washed twice before aging except those marked b which were washed once only. Zero-time samples were stored at 0°C during the reactivation period. Spontaneous reactivation was calculated as percentage increase in residual enzyme activity during 18 hr at 37°C. a Incubation for 18 hr at pH 7.0 instead of 8.0.
was as effective as KF in reactivating NTE after inhibition by leptophos oxon [experiment not shown but carried out as in the original study with that compound (15)] and did not adversely affect control uninhibited NTE.
After inhibition by (R)r( +) isomers, NTE was reactivated by INAP to within I6% of control levels immediately after inhibition (six experiments) and there was no decrease in reactivatibility and, therefore, no aging after a further 18 hr at 37°C and
PHENYLPHOSPHONATEISOMERS
pH 8.0 (three experiments). For (R)r( + )inhibited AChE, reactivatibility [mean + SD (n)] declined during 18 hr at pH 8.0 and 37°C from 86 + 7% (6) of zero-time control to 66 c 7% (4) of the 18-hr control. Aging was therefore 23% and two experiments detected 12% aging at pH 7.0. After inhibition of either NTE or AChE by (S),( -> isomers reactivation by INAP was very slight (O11%) in numerous experiments even at zero-time so that no conclusion about aging could be drawn. The failure to reactivate may have been due to steric hindrance of access of the reactivator molecule to the target bond. However an alternative possibility was considered as described below. Further Study of Aging of Enzymes after Inhibition by (S),( - ) Isomers
Because these isomers were not potent inhibitors and also their physical solubility in buffer was limited to about 200 p.M it was necessary to incubate enzyme with inhibitor for periods of 30 or 60 min to obtain reasonable inhibition of AChE and NTE, respectively. Since we know that some inhibited NTEs age with tii, of l-5 min (15) it seemed possible that aging of NTE (and possibly also of AChE) was virtually completed during the inhibition period so that only oxime-resistant inhibited enzyme encountered the reactivator. To investigate this possibility we adopted an indirect approach using the following reasoning: (i) The contrast in response to INAP after inhibition by the chiral isomers demonstrates that distinct inhibited species were formed in the two cases. Inhibition may have proceeded with either retention or inversion of the optically active form of the ethyl phenylphosphonyl moiety at the active site, but, clearly, the product of inhibition was not a mixture of both forms as would be expected if inhibition proceeds with racemization. (ii) Racemic EPNO is active against NTE with I,, about 2 @4 for 20 min at 37°C [Ref (14) and Lotti and Johnson, unpublished]. Since EPNO differs from MCB and DCB only in the nature of its
ANDNTE
139
most labile group (6nitrophenyl instead of S-halobenzylthiolo), inhibition by EPNO would be expected to form ethyl phenylphosphonyl-NTE indistinguishable chemically from the product of reaction of MCB or DCB with NTE. (iii) It is likely that each isomer present in a racemic mixture of EPNO would react with target enzymes independently and at rates largely unaffected by the presence of the other enantiomer: though likely this is not certain but it is not vital to the argument. (iv) Rough measures of inhibitory power of the part-purified individual stereoisomers of EPNO were obtained in this lab some years ago (Lotti and Johnson unpublished) and these agree with the report by Ohkawa et al. (14): inhibition of NTE by the L( -) isomer was favored over D( + ) by a factor of i S-2 and inhibition of AChE by the D( +) was 3-4 x faster than inhibition by L( -). With the above statements in mind we therefore inhibited NTE or AChE in separate experiments by incubation with a calculated concentration of racemic EPNO for 1 min at 37°C and then quenched both inhibition and aging by diluting the tissue + 19 vol of ice-cold pH 8.0 buffer. Reactivation of inhibited enzyme was attempted immediately thereafter with the usual procedures. Table 3 shows that treatment of this EPNO-inhibited NTE with INAP caused 40-57% reactivation at zero time but lesser responses declining to a plateau of 17-23% as the delay before addition of INAP was increased through 1 to 18 and 60 min. It appears, therefore, that 60 min after inhibition of NTE by racemic EPNO two forms of inhibited enzyme were present with characteristics the same as those of NTE inhibited by (R)p( +) and (S),( - ) forms of MCB and DCB: the one which could not be reactivated had undoubtedly aged. The aged form was predominant and therefore was probably derived from the more potent (-)-EPNO isomer. We conclude that the inhibited NTE obtained during 60 min incubations with the (S),( -) form of MCB or DCB had also aged and that this was the
140
JOHNSON, TABLE
Reactivation Delay before treatment with INAP (min)
READ,
3
by INAP of NTE after I-min Irihibition by Racemic EPNO Activity
after treatment
No INAP (a)%
+ INAP @)%
Reactivation &+J%,
0
5 10 9
57 61 4.5
55 57 40
1
2
30 32 33
z
31 35 31
18
3
24
22
60
2 4
19 26
17 23
Note. Brain particles were incubated with racemic EPNO (400 PM) for 1 min; inhibition was halted by dilution, and aging was allowed to proceed for various times before reactivation with INAP was attempted as described under Materials and Methods. Activities are given as percentage activity of a control treated exactly as above but “inhibited” with solvent but without EPNO. Recovery of INAP-treated controls was 100 f 1% (SD, n = 9).
reason for lack of success in the reactivation experiments with these isomers. In the experiment with l-min inhibition of AChE by racemic EPNO (5 p.M) at least 95% reactivation was obtained at four times from 0 to 50 min afterward. Since the amount of oxime-resistant inhibited activity was so small we presume that inhibition by the (+)-isomer was dominant and the procedure could not be exploited to study possible aging of the (-)-inhibited form. DISCUSSION
This study has revealed further contrasts at the molecular level between AChE and NTE which are the targets, respectively, for the acute and the delayed toxic effects of OP esters. The stereoselectivity of the enzymes for these inhibitors differ. Thus the (R)p( + ) isomers are more inhibitory to NTE while (S),( -) are more inhibitory to AChE. It is interesting to note that for another phosphonothiolate, Fonofos oxon (ethyl S-phenyl ethylphosphonothiolate), the (S),( -)-isomer was reported to be more inhibitory than (R)p( +) against several cholinesterases, trypsin, chymotrypsin, serum and liver esterases, and overall brain hy-
AND
YOSHIKAWA
drolytic activity against aryl acetates (19). However, bulk activity of brain against aryl butyrate or valerate was about equally sensitive to both isomers: NTE probably represents about U-20% of this activity so it is quite possible that NTE is actually more sensitive to (R)p( +)-fonofos oxon thereby pulling the ratio of bulk brain esterase sensitivities to the two isomers toward equality. Since the reactivity of enantiomers in solution is, presumably, identical, the differing inhibitory power of stereoisomers against AChE and NTE must be a function of goodness-of-fit to the respective active sites. Steric factors are important also in the reactions subsequent to inhibition and, again, the enzymes differ. Table 2 shows that neither inhibited form of AChE undergoes measurable spontaneous reactivation in 18 hr at pH 8.0 but the (R)p( -)-inhibited NTE does. There is great contrast between aging behaviour of the (R)p( + )-inhibited NTE (t’h = few minutes) and the (S),(-) form (no aging in I8 hr). We cannot say whether the (S),( -) form of inhibited AChE ages rapidly or is refractory to oxime treatment for some other reason. The (R)p( +) form did age slightly in 18 hr at pH 8.0 or 7.0. No test was made of effect of change of pH on aging of inhibited NTE but it is known that there was little effect after inhibition by some other phosphonates (13. Predictions about comparative acute or delayed effects in vivo can often be made after studies such as are reported here of in vitro interactions of OP direct inhibitors with the target enzymes (4,9>. It is not possible to predict what dose will lead to high inhibition of AChE and acute toxic effects since distribution of the agent in the body, metabolic disposal, and excretion are dominant factors. However, the rule seems fairly vindicated that a neuropathic dose will be less than a lethal dose only if the potency of the proximal toxin against NTE is greater than that against AChE, i.e., k aNTE is greater than kaAChE. On this basis
PHENYLPHOSPHONATE
and using the ratios from Table 1 we might predict that doses of the (S),( -) isomers many times the unprotected LD,, would be required to cause high in viwo inhibition of NTE with consequent initiation of neuropathy while for the (R)r( +) isomers doses only 2-3 x LD,, given to hens with concomitant prophylaxis and therapy against acute toxicity should be sufficient to cause high inhibition of NTE. However, such prediction of responses in vivo are totally dependent on the assumption that the same chemicals are being studied in vitro and in vim.
This study has revealed that the ethyl phenylphosphonylated NTE obtained from (R)r( +)-DCB or MCB does not age significantly in vitro. The question arises as to what would happen if 100% of NTE were converted to the same steric form in vivo: would there be a neuropathic response or would birds become protected as they do when the NTE is inhibited to a non-ageable phosphinylated form (4, 5)? Delayed neuropathy and NTE studies have been performed with three pesticides with different leaving groups but which would each be expected to give ethyl phenylphosphonylated NTE. Single doses of racemic EPN, ethyl leptophos, or cyanofenphos each cause delayed neuropathy associated with high inhibition of NTE (12, 14, 20-22). However, for the latter two compounds the measured level of NTE inhibition required to initiate neuropathy seemed to be 90-100% rather than the usual level of 70-80% as found for many compounds (7). Some of these authors (20-22) suggested that, for these pesticides, some of the inhibited NTE might be in a slow-aging configuration and the present study lends credibility to that suggestion. Some work has been done in vivo with resolved isomers of EPN but not, unfortunately, with EPNO. A single dose of L( - ) EPN can cause neuropathy (12) and high inhibition of NTE was found, as expected, l-2 days after dosing (14). For D( +)-EPN single doses up to 89 mg/kg did not cause neuropathy (12) and in one bird
ISOMERS
AND NTE
141
the NTE inhibition after 50 mglkg was only 50% which is well below the threshold for initiation (14). No aging measurements were made in these studies. Subchronic feeding of hens by either stereoisomer of EPN initiated neuropathy when the dose x time was extended sufficiently (13). To clarify interpretation of the molecular events of initiation by these compounds it would be necessary to know (i) the degree of aging (if any) and the clinical response to a highly inhibitory single dose of D( +)EPN, (ii) what were the accumulated masses of inhibited NTE under subchronic regimes which enabled both isomers to cause equal clinical effect, (iii) to what extent aging had actually occurred and (iv) whether conversion of D( + )-EPN to EPNO was totally stereospecific or whether EPN underwent significant racemisation in rive in the same way as has been shown for fonofos isomers (23). Studies in viva with the easily synthesized direct-acting stereoisomers of MCB and DCB may prove very useful in a closer examination of the intimate details of the neuropathy initiation process. REFERENCES 1. M. K. Johnson, A phosphorylation site in brain and the delayed neurotoxic effect of some organophosphorus compounds. Biochem. J. 111, 487 (1969a). 2. M. K. Johnson, The delayed neurotoxic effect of some organophosphorus compounds. Identification of the phosphorylation site as an esterase, Biachem. J. 114, 711 (1969b). 3. M. K. Johnson, Initiation of organophosphate neurotoxicity, Toxicol. Appl. Pharmacol. 61, 480 (1981). 4. M. K. Johnson, The target for initiation of delayed neurotoxicity by organophosphorus esters: biochemical studies and toxicological applications. in “Rev. Biochem. Toxicol.” (E. Hodgson, J, R. Bend, and R. M. Philpot, Eds. ). p. 141 Elsevier, New York (1982). 5. M. K. Johnson, The primary biochemical lesion leading to the delayed neurotoxic effects of some organophosphorus esters, J. Neurochem. 23, 785 (1974). 6. B. Clothier and M. K. Johnson, Rapid aging of neurotoxic esterase after inhibition by diiso-
142
7.
8.
9.
10.
11.
12.
13.
14.
JOHNSON,
READ,
propyl phosphorofluoridate, Biochem. J. 177, 549 (1979). M. K. Johnson, Organophosphorus esters causing delayed neurotoxic effects: Mechanism of action and structure/activity studies, Arch. Toxicol. 34, 259 (1975a). M. K. Johnson, Structure-activity relationships for substrates and inhibitors of hen brain neurotoxic esterase. Biochem. Pharmacol. 24, 797 (1975c). M. Lotti and M. K. Johnson, Neurotoxicity of organophosphorus pesticides: Predictions can be based on in vitro studies with hen and human enzymes, Arch. Toxicol. 41, 215 (1978). H. P Benschop, C. A. G. Konings, J. Van Genderen, and L. P. A. De Jong. Isolation, anticholinesterase properties and acute toxicity of the four stereoisomers of the nerve agent soman, Toxicol. Appl. Pharmacol. 72, 61 (1984). M. K. Johnson, D. J. Read, and H. P. Benschop, Interaction of the four stereoisomers of soman with acetylcholinesterase and neuropathy target esterase of hen brain, Biochem. Pharmacol., 34, 1945 (1985). H. Ohkawa, N. Mikami, Y. Okuno, and J, Miyamoto, Stereospeciticity in toxicity of the optical isomers of EPN, Bull. Environ. Contam. Toxicol. 18, 534 (1977). M. B. Abou-Donia, D. G. Graham, A. A. Komeil, A. A. Nomeir, and W. C. Dauterman, Stereospecificity of cholinergic and delayed neurotoxic effects of O-ethyl 0-Cnitrophenyl phenylphosphonothioate optical isomers: Effect of daily oral administration to hens, in “Advances in Neurotoxicology” (L. Manzo, Ed.), p. 237 Pergamon, Oxford (1980). H. Ohkawa, H. Oshita, and J. Miyamato, Comparison of inhibitory activity of various organophosphorus compounds against acetylcholinesterase and neurotoxic esterase of hens with
AND YOSHIKAWA
15.
16.
17.
18. 19.
20.
21.
22.
23.
respect to delayed neurotoxicity, Biochem. Pharmacol. 29, 2721 (1980). B. Clothier and M. K. Johnson, Reactivation and aging of neurotoxic esterase inhibited by a variety of organophosphorus esters, Biochem. J. 185, 739 (1980). H. Yoshikawa, T. Shono, and M. Eto, A facile synthesis and insecticidal activity of optically active phenylphosphonothiolates, J. Pestic. Sci. 9, 455-462 (1984). B. Clothier, M. K. Johnson, and E. Reiner, Interaction of some trialkylphosphorothiolates with acetylcholinesterase: Characterisation of inhibition, aging and reactivation, Biochim. Biophys. Acta. 660, 306 (1981). B. Clothier, “Reactivation and aging of esterases,” M. Phil. Thesis. Council for Nat. Academ. Awards, London (1979). P. W. Lee, R. Allahyari, and T. R. Fukuto, Studies on the chiral isomers of Fonofos and Fonofos oxon. 1. Toxicity and antiesterase activity. Pestic. Biochem. Physiol. 8, 146 (1978). H. H. Reinders, L. G. Hansen, R. L. Metcalf, and R. A. Metcalf. In vitro and in vivo inhibition of chicken brain neurotoxic esterase by leptophos analogs, Pestic. Biochem. Physiol. 28, 67 (1983). L. G. Hansen, E. M. Cozzi, R. L. Metcalf, and T. K. Hansen, Effect of disposition on the relative delayed neurotoxicity potency among leptophos analogues. Pestic. Biochem. Physiol.. in press. N. S. Ahmed, K. S. El-Gendy, J. D. Farmer, R. Novak, N. Bakry, and S. A. Soliman, Timecourse and dose-dependent in vivo effect of ethyl-leptophos on hen brain neurotoxic esterase activity, Toxicol. Let?. 18 (Suppl. I), 111 (1983). P. W. Lee, R. Allahyari, and T. R. Fukuto, Studies on the chiral isomers of Fonofos and Fonofos oxon. II. In vitro metabolism, Pestic. Biochem. Physiol. 8, 158 (1978).
BIOCHEMISTRY
AND
PHYSIOLOGY
25, 133- 142 (1986)
The Effect of Steric Factors on the Interaction of Some Phenylphosphonates with Acetylcholinesterase and Neuropathy Target Esterase of Hen Brain M. K. JOHNSON,]
D. J. READ,
AND H. YOSHIKAWA*
Molecular Toxicology Section, Toxicology Unit MRC Laboratories. Woodmansterne Road. Carshulton. Surrey, SM5 4EF, England; and *Deportment of Environmental Chemistry. Furldry ofEngineering, Kyushu Kyoritstt University, Kitukyushrt 807. Japan
Received January 14. 1985; accepted April 12, 1985 At 37°C and pH 8.0 the resolved isomers of S-(4-chlorobenzyl or 2.4-dichlorobenzyl) ethyl phenylphosphonothiolate are moderate inhibitors of AChE of hen brain (k, = 300-1400 M-‘min-‘) and weak inhibitors of neuropathy target esterase (NTE) (k, = 40-240 M-‘min-I). The two (R),( +) isomers are more active against NTE than are the (S),( - ) while the opposite is true for AChE. NTE inhibited by either (R),( + ) isomer underwent slow spontaneous reactivation (about 30% in 18 hr) but (S),( - )-inhibited NTE did not reactivate. No spontaneous reactivation was detected for AChE inhibited by either steric form. AChE inhibited by (R)p( + ) isomers could be reactivated by N-methylpyridinium-2-aldoxime methanesulfonate and slow aging was detected (23% in 18 hr). NTE inhibited by (R)p( + ) isomers could be reactivated by o-isonitrosoacetophenone and no significant aging occurred in 18 hr at 37°C. Neither of the enzymes could be reactivated 60 min after commencement of inhibition with saturated solutions of the (S)p( -) isomers. NTE inhibited by racemic EPNO in a I-min incubation formed a mixture of rapidly aging (tic = I min) and apparently non-aging inhibited NTE. Since EPNO should form the same ethyl phenylphosphonylated enzymes as the isomers under study we conclude that the oxime-resistance of inhibited NTE after inhibition by these (S)p( - )-isomers is due to rapid formation of aged enzyme during the (necessarily) long inhibition period. Substantial inhibition of NTE in viva should be possible in hens protected against cholinergic effects of a dose of either (R),( + ) isomer. The toxicological consequence of such inhibition (? neuropathy or protection) should be investigated. %’ 1986 Academic Press, Inc.
INTRODUCTION
Two major toxic effects of OP* esters are known. These are the acute toxicity initiated by covalent organophosphylation of i To whom correspondence should be addressed. * Abbreviations used: OP, organophosphorus; AChE, acetylcholinesterase; NTE, neuropathy target esterase, formerly called neurotoxic esterase; INAP, w-isonitrosoacetophenone; P2S, N-methylpyridinium 2-aldoxime methanesulphonate; MCB, ethyl S-[4chlorobenzyl] phenylphosphonothiolate; DCB. ethyl S-[2,4-dichlorobenzyl] phenylphosphonothiolate; EPNO (the oxon of the pesticide EPN) ethyl 4-nitrophenyl phenylphosphonate.
the active centre of AChE and delayed neuropathy initiated by a similar reaction on the active site of NTE (l-4). For delayed neuropathy a second essential step in the initiation process is aging of the inhibited enzyme (Reaction (4) in Scheme 1) (5, 6). The structural features in OP esters associated with inhibitory power against AChE and NTE are different (7-9) so that some anticholinesterase compounds are known which are neuropathic below LD,, while others are not neuropathic in animals even at doses far above unprotected LD,, given in conjunction with prophylaxis and therapy against acute toxicity.
133 0048-3575/86 $3.00 Copyright 0 1986 by Academic Press. Inc All rights of reproduction in any form reserved
134
JOHNSON,
O
E OH+.-!’
SCHEME 1. Steps Michaelis complex; (4) aging.
OR
(1)
[
READ,
AND
0 II/
EoH---x-P,~~
in the interaction (2) phosphorylation
OR
YOSHIKAWA
1
*
(2)
0
--‘Eo-P;
OR I
of an esterase with an organophosphorus of enzyme; (3) reactivation (spontaneous
Many organophosphonates of interest as pesticides or potential chemical warfare agents contain a chiral centre and in some cases the resolved isomers have markedly different toxicities. Thus the two P( -) isomers of soman have 10-100x the acute toxicity to mice of the P( +) isomers while in vitro the anticholinesterase activities differ by at least 1000x (10). Against NTE in vitro the potency of these four isomers differed over a lo-fold range (11). In vivo diffc :nces in neuropathic potency have been reported for isomers of the pesticide EPN (12, 13) and the difference has been attributed to the greater anti-NTE potency of the ( -) isomer of EPNO (14). Only one study of aging of inhibited NTE after inhibition by resolved isomers has been reported (I 1). This was with isomers of soman and we were surprised to find that some structures exist which yielded forms of inhibited NTE which aged slowly: for most of the inhibitory compounds (nonchiral phosphates or racemic mixtures of phosphonates) which had been reported previously, aging of inhibited NTE was very fast (15). Since aging of inhibited.NTE is an essential second step in the initiation of delayed neuropathy, further studies of aging of inhibited NTE are of considerable interest to toxicologists as well as to enzymologists. Some years ago Professor W. Dauterman
OR
”!
inhibitor. orforced
(I) Formation of by oximes or KF);
kindly supplied us with small samples of resolved isomers of EPNO. However, some decomposition had occurred during transshipment and we were unable to do more than preliminary measures of I,, values which agreed well with those subsequently published by Ohkawa et al. (14). However, the resolution of ethyl phenylphosphonothiolic acid and formation of S-esters with retention of configuration is now well established and relatively easy to perform. Studies on anti-cholinesterase and insecticidal activity of such resolved chiral esters have been reported (16). Although the thioloester bond is not a favored structural feature for NTE inhibition, the phenylphosphonate moiety is (7). From a wide range of available structures we therefore chose the resolved isomers of S-4-monochlorobenzyl and S-2,Cdichlorobenzyl esters as potential inhibitors for study. On the basis of general chemistry and of proof with one S-methyl phosphorothiolate (17) it is reasonable to predict that these compounds should form exactly the same ethyl phenylphosphonylated esterases in vitro as would be obtained when inhibiting with the oxons of EPN, cyanofenphos or ethyl leptophos . We report here on the inhibition and aging of hen brain AChE and NTE using these isomers and seek to interpret the significance for effects in vivo.
PHENYLPHOSPHONATE MATERIALS
AND
METHODS
Chemicals The resolved and purified [(R)r( +) and (S),( - )] isomers of MCB and DCB were prepared in Japan as described previously (16), shipped by air to the United Kingdom and stored in desiccators at -5°C until required: the dry compounds are very stable under these conditions and optical purity was greater than 98%. Stock solutions in dry acetone were prepared as required. The unresolved oxon of EPN was a gift of Dr. H. Ohkawa, Sumitomo Chemical Company: it was essentially free of 4-nitrophenol. INAP was from Aldrich Chemical Company and melted at 127°C (lit 126128°C); an older sample melted over a wide range and, while it appeared active as a reactivator, it was not used for definitive experiments. P2S was a gift from Chemical Defense Establishment, Porton Down. Buffers. For AChE assay solutions of NaH?PO, and Na,HPO, (each 100 mA4) were mixed to give the desired pH. For most other experiments Tris (50 mM)/ EDTA (0.2 mM) was adjusted to pH 8.0 with HCI (about 12 M) at ambient temperature. To prepare the reactivation medium for NTE 1 vol of INAP (500 mM in acetone) was added to 24 vol Tris base (50 mM)l EDTA (0.2 m&f) at 37°C and the pH was adjusted to 9.0 with HCl: the INAP acts as a buffer in the pH range 8-9 and is probably a more effective reactivator in its ionized form. Inhibition, Aging, Reactivation, and Assay of NTE Detailed protocols are given in Ref. (11) and brief comments are given here. The activity of NTE is normally measured as the difference in phenyl valerate esterase activity between two samples which have been preincubated with a non-neuropathic progressive inhibitor (paraoxon or benzenesulphonyl fluoride) with or without the neuropathic inhibitor, mipafox. The rea-
ISOMERS
AND
NTE
135
sons for the choice of inhibitors used in aging studies were presented in Refs. (6) and (15). We obtained improved dispersion of sedimented particles prior to assay by including Triton X-100 (0.01%) in the final dispersion medium as described recently (11). (a) Study of time course of inhibition. Paired tissue samples, routinely called “P” and “M,” were prepared and used exactly as described (11, 6); the samples are identical except that P contains active NTE while M does not. Inhibitors were added from concentrated stock solutions in acetone and the final solvent concentration was not more than 1% (v/v). (b) Study of spontaneous reactivation and aging. (i) For the situation when the aging period was more than 1 hr the improved procedure of Ref. (11) was used in which inhibited enzyme was separated from unreacted inhibitor by sedimentation and resuspension in fresh buffer before the commencement of the timed aging period. In initial experiments a confusing amount of ongoing inhibition occurred during the 18-hr aging period at 37°C. Thereafter the inhibited particles were washed by one or two cycles of resedimentation and resuspension. (ii) For the cases when aging was rapid and the inhibition period limited to a few minutes the procedure of Ref. (6) was used except that the first dilution step ( x 20) after a 1-min inhibition used ice-cold pH 8.0 Tris buffer to halt the progress of inhibition more effectively during the 30set delay before a further 4.1 x dilution into the reactivation medium at 37°C. The nucleophilic reactivator was INAP and not KF as used previously: inhibited tissue (8 ml in pH 8 buffer) was mixed with 25 ml of a solution of INAP [20 mM in acetone/pH 9 buffer (4:96 v/v)] and incubated at 37°C for 30 min prior to cooling and sedimentation of particles etc. as before (11). The high concentration of acetone was necessary to prevent crystallization of INAP during the cold centrifugation.
JOHNSON,
136
READ,
AND
Inhibition, Aging, and Reactivation of AChE Inhibition and Ellman assay procedures were as fully described before (11). Manipulation of tissue for aging and reactivation was exactly as for NTE except that P and M samples were not needed. The reactivation procedure used P2S (1 mM final at pH 8.0 for 20 min at 37°C).
YOSHIKAWA AChE
NTE
2.0 1.9 1.8 1.7 1.6 1.5 1.4
RESULTS
Inhibition of Enzymes As observed by eye the solubility limit of the inhibitors in buffer at 37°C was about 200 pJ4. Figure 1 shows semi-log plots of enzyme activity vs time of preincubation with inhibitor. These were linear and the first-order rate constants derived from the slopes of the lines were approximately proportional to inhibitor concentration up to the solubility limit. Against NTE the (S)p( -) isomers were so weak that even at 100-200 PM the slopes of the first order plots were very shallow. The second order rate constant for inhibition (k,) was derived from the slope (k’) of each line in Fig. 1 using the relationship: k, =
k’
-2.303
[Cl
where [Zj is the molar concentration of inhibitor. Mean values are given in Table 1 which shows that in each case the compounds were more active against AChE than against NTE. Against AChE the (S),( -) isomers were 2-3 x more active than were the (R)p(+) isomers but against NTE (R)r( +) isomers were more active than (S),( -). It follows that the ratios k NTEIkaAChEdiverged greatly being about 0:4 for (R)r(+) isomers and 0.03-0.09 for W,( - 1. Spontaneous Reactivation of Inhibited NTE and AChE Table 2 shows that there was no evidence for spontaneous reactivation of NTE inhibited by (S),( -) isomers: activity at 18 hr
.p 1.7 .c
1.6
E
1.5
1.9 1.8 1.7
MC8 SIpI
1.6 1.5 1.4
t....
10
Inhibition
Time
20
30
40
(min)
FIG. 1. Semi-lag plots of progressive inhibition of hen brain AChE and NTE by isomers at concentrations shown and at pH 8.0 and 37°C. For AChE residual activity was determined after preincubation of tissue with inhibitor for periods up to 40 min. For NTE paired tissue samples differing only by containing active or inactivated NTE were prepared as described under Materials and Methods. These paired samples were further preincubated with test inhibitor for periods up to 40 min and the residual phenyl valerate esterase activity was measured and the NTE activity calculated by difference. Slopes (k’) of the progress lines were measured and used to derive k, values given in Table 1. Each value of k’ was obtained from a line through 4-6 points: the error in these values of k’ were much smaller than the error of the mean value of k, derived from each family of lines.
PHENYLPHOSPHONATE
ISOMERS
TABLE Second
Order
Rate Constants (k,) Phenylphosphonate
137
AND NTE
1
(M-l min-‘) for Stereoisomers
Inhibition at 37°C.
pH
of AChE 8.0
and
NTE
by
Isomer Enzyme
DCB-(R),( + )
MCB-(R),( + )
DCB-(S),( - )
MCB-(S),( - 1
AChE NTE Approximate ratio of rate constants
560 (3) 240 (3)
300 (4) 120 (3)
1400 (4) 40 (3)
I100 (4) 95 (2)
0.4
0.4
0.03
0.09
k NTE//( AChE a a
Nofe. Mean values of k, were derived from the slopes of lines in Fig 1. Individual values differed from mean by less than 10% except for MCB-(R),(+) against NTE where the range from mean was 16%. The number of determinations is given in parentheses:
was less than that at zero time even when allowance was made for some control losses due to instability. We presume that sufficient traces of lipophilic inhibitor survived the initial sedimentation and washing of inhibited particles to cause further inhibition. For this reason it is impossible to say that no spontaneous reactivation occurred but the results are in clear contrast to those with (R)r( +) isomers where activity increased during 18 hr in spite of the control losses and the possibility of continued inhibition at concentrations similar to those of the (S),( -) isomers. The calculated percentage values for spontaneous reactivation are very crude since they depend on a ratio derived from comparatively small differences between large numbers. However, the effect is sufficiently reproducible to be considered real. For AChE no spontaneous reactivation was detected after inhibition by either isomer. The initial experiments were done in conjunction with aging studies and inhibition was at high concentrations for short times. Latterly we extended inhibition time up to 6 hr to use lower concentration of inhibitor: even this procedure followed by sedimentation of the particles and two resuspension-washes with cold buffer did not entirely eliminate the problem of ongoing inhibition during the 18-hr incubation so that the failure to detect reactivation only shows that the rate. if any, is very low.
Znduced Reactivation and Aging of Inhibited NTE and AChE Most previous studies of aging of inhibited NTE have used KF as reactivator since there is unlikely to be steric hindrance to access of this reactivator to the active site and also the agent is both cheap and easily removed by sedimentation of the particlebound enzyme. Preliminary experiments on reactivation by KF of either NTE or AChE of hen brain after inhibition by the phenylphosphonothiolates showed only massive increases in inhibition. This we attribute to the conversion of residual amounts of the comparatively poor inhibitors to analogous fluoridates which are likely to be much more potent inhibitors: a similar problem has been found previously with transformation of paraoxon and some other nitrophenyl esters in contact with KF (15). We therefore used the well-known P2S instead of KF as AChE reactivator (see Materials and Methods). Clothier (18) found that a saturated solution of INAP, (about 5 mM) was a moderately effective reactivator of diethylphosphinyl-NTE. We have now found that higher concentrations of INAP remain in solution throughout the reactivation and cold centrifugation steps if the medium is made up to pH 9.0 and contains acetone (3% v/v) as described under Materials and Methods section. Incubation for 30 min at 37°C in this reactivation medium
JOHNSON,
138
READ, AND YOSHIKAWA TABLE 2
Spontaneous
Reactivation
of Inhibited
NTE
and AChE
Activity at zero time Inhibition cone (PM)/ time (min)
Isomer NTE DCB-(R),( + 1
(a) Control uninhibited (%I
(b) Inhibited (%o)
during
18 hr at 3X,
pH 8.0
Activity at 18 hr (cl Control (%I
Cd) Inhibited (So)
Spontaneous reactivation dlc
-
bla
%
100 - b
250/60b 250/60b 100/180 601270
100 100 100 100
25 32 14 49
80 74 84 83
34 36 24 53
24 25 17 29
MCB-(R),( + 1
300/60b 100/180 801270
100 100 100
35 38 39
74 84 83
42 55 56
34 44 47
DCB-(S),( - )
800/6Ob 200/180 100/360
100 100 100
56 55 56
80 84 89
22 35 44
-64 -29 -15
MCB-(S),( - 1
800/60b 200/180 100/360
loo loo 100
76 60 56
80 84 89
31 43 46
-154 -23 - 10
+1
500125’ 500125 601270 30/200 201360
100 100 100 100 100
9 9 14 40 41
71 87” 92 96 91
2 70 7 18 23
-7 -la -35 -7 -27
+1
5OOl25’ 500125 801270 50/200 251360
100 loo 100 loo loo
14 11 11 23 34
71 87” 92 96 91
4 15” 12 23 31
-10
- 1
300/2Sb 15/200 201360
100 100 100
36 73 47
69 96 91
3 31 19
-50 -150 -49
MCB-(S),( - )
300125’ 300125 20/200 151350
100 100 100 100
20 21 48 48
69 87“ 96 91
4 13” 34 35
AChE DCWV,(
MCWR),(
DCB-W,(
7” 2 1 0
-18 - 8” -24 -18
Note. Procedure was as described under Materials and Methods: all inhibited and “solvent-inhibited” control particles were washed twice before aging except those marked b which were washed once only. Zero-time samples were stored at 0°C during the reactivation period. Spontaneous reactivation was calculated as percentage increase in residual enzyme activity during 18 hr at 37°C. a Incubation for 18 hr at pH 7.0 instead of 8.0.
was as effective as KF in reactivating NTE after inhibition by leptophos oxon [experiment not shown but carried out as in the original study with that compound (15)] and did not adversely affect control uninhibited NTE.
After inhibition by (R)r( +) isomers, NTE was reactivated by INAP to within I6% of control levels immediately after inhibition (six experiments) and there was no decrease in reactivatibility and, therefore, no aging after a further 18 hr at 37°C and
PHENYLPHOSPHONATEISOMERS
pH 8.0 (three experiments). For (R)r( + )inhibited AChE, reactivatibility [mean + SD (n)] declined during 18 hr at pH 8.0 and 37°C from 86 + 7% (6) of zero-time control to 66 c 7% (4) of the 18-hr control. Aging was therefore 23% and two experiments detected 12% aging at pH 7.0. After inhibition of either NTE or AChE by (S),( -> isomers reactivation by INAP was very slight (O11%) in numerous experiments even at zero-time so that no conclusion about aging could be drawn. The failure to reactivate may have been due to steric hindrance of access of the reactivator molecule to the target bond. However an alternative possibility was considered as described below. Further Study of Aging of Enzymes after Inhibition by (S),( - ) Isomers
Because these isomers were not potent inhibitors and also their physical solubility in buffer was limited to about 200 p.M it was necessary to incubate enzyme with inhibitor for periods of 30 or 60 min to obtain reasonable inhibition of AChE and NTE, respectively. Since we know that some inhibited NTEs age with tii, of l-5 min (15) it seemed possible that aging of NTE (and possibly also of AChE) was virtually completed during the inhibition period so that only oxime-resistant inhibited enzyme encountered the reactivator. To investigate this possibility we adopted an indirect approach using the following reasoning: (i) The contrast in response to INAP after inhibition by the chiral isomers demonstrates that distinct inhibited species were formed in the two cases. Inhibition may have proceeded with either retention or inversion of the optically active form of the ethyl phenylphosphonyl moiety at the active site, but, clearly, the product of inhibition was not a mixture of both forms as would be expected if inhibition proceeds with racemization. (ii) Racemic EPNO is active against NTE with I,, about 2 @4 for 20 min at 37°C [Ref (14) and Lotti and Johnson, unpublished]. Since EPNO differs from MCB and DCB only in the nature of its
ANDNTE
139
most labile group (6nitrophenyl instead of S-halobenzylthiolo), inhibition by EPNO would be expected to form ethyl phenylphosphonyl-NTE indistinguishable chemically from the product of reaction of MCB or DCB with NTE. (iii) It is likely that each isomer present in a racemic mixture of EPNO would react with target enzymes independently and at rates largely unaffected by the presence of the other enantiomer: though likely this is not certain but it is not vital to the argument. (iv) Rough measures of inhibitory power of the part-purified individual stereoisomers of EPNO were obtained in this lab some years ago (Lotti and Johnson unpublished) and these agree with the report by Ohkawa et al. (14): inhibition of NTE by the L( -) isomer was favored over D( + ) by a factor of i S-2 and inhibition of AChE by the D( +) was 3-4 x faster than inhibition by L( -). With the above statements in mind we therefore inhibited NTE or AChE in separate experiments by incubation with a calculated concentration of racemic EPNO for 1 min at 37°C and then quenched both inhibition and aging by diluting the tissue + 19 vol of ice-cold pH 8.0 buffer. Reactivation of inhibited enzyme was attempted immediately thereafter with the usual procedures. Table 3 shows that treatment of this EPNO-inhibited NTE with INAP caused 40-57% reactivation at zero time but lesser responses declining to a plateau of 17-23% as the delay before addition of INAP was increased through 1 to 18 and 60 min. It appears, therefore, that 60 min after inhibition of NTE by racemic EPNO two forms of inhibited enzyme were present with characteristics the same as those of NTE inhibited by (R)p( +) and (S),( - ) forms of MCB and DCB: the one which could not be reactivated had undoubtedly aged. The aged form was predominant and therefore was probably derived from the more potent (-)-EPNO isomer. We conclude that the inhibited NTE obtained during 60 min incubations with the (S),( -) form of MCB or DCB had also aged and that this was the
140
JOHNSON, TABLE
Reactivation Delay before treatment with INAP (min)
READ,
3
by INAP of NTE after I-min Irihibition by Racemic EPNO Activity
after treatment
No INAP (a)%
+ INAP @)%
Reactivation &+J%,
0
5 10 9
57 61 4.5
55 57 40
1
2
30 32 33
z
31 35 31
18
3
24
22
60
2 4
19 26
17 23
Note. Brain particles were incubated with racemic EPNO (400 PM) for 1 min; inhibition was halted by dilution, and aging was allowed to proceed for various times before reactivation with INAP was attempted as described under Materials and Methods. Activities are given as percentage activity of a control treated exactly as above but “inhibited” with solvent but without EPNO. Recovery of INAP-treated controls was 100 f 1% (SD, n = 9).
reason for lack of success in the reactivation experiments with these isomers. In the experiment with l-min inhibition of AChE by racemic EPNO (5 p.M) at least 95% reactivation was obtained at four times from 0 to 50 min afterward. Since the amount of oxime-resistant inhibited activity was so small we presume that inhibition by the (+)-isomer was dominant and the procedure could not be exploited to study possible aging of the (-)-inhibited form. DISCUSSION
This study has revealed further contrasts at the molecular level between AChE and NTE which are the targets, respectively, for the acute and the delayed toxic effects of OP esters. The stereoselectivity of the enzymes for these inhibitors differ. Thus the (R)p( + ) isomers are more inhibitory to NTE while (S),( -) are more inhibitory to AChE. It is interesting to note that for another phosphonothiolate, Fonofos oxon (ethyl S-phenyl ethylphosphonothiolate), the (S),( -)-isomer was reported to be more inhibitory than (R)p( +) against several cholinesterases, trypsin, chymotrypsin, serum and liver esterases, and overall brain hy-
AND
YOSHIKAWA
drolytic activity against aryl acetates (19). However, bulk activity of brain against aryl butyrate or valerate was about equally sensitive to both isomers: NTE probably represents about U-20% of this activity so it is quite possible that NTE is actually more sensitive to (R)p( +)-fonofos oxon thereby pulling the ratio of bulk brain esterase sensitivities to the two isomers toward equality. Since the reactivity of enantiomers in solution is, presumably, identical, the differing inhibitory power of stereoisomers against AChE and NTE must be a function of goodness-of-fit to the respective active sites. Steric factors are important also in the reactions subsequent to inhibition and, again, the enzymes differ. Table 2 shows that neither inhibited form of AChE undergoes measurable spontaneous reactivation in 18 hr at pH 8.0 but the (R)p( -)-inhibited NTE does. There is great contrast between aging behaviour of the (R)p( + )-inhibited NTE (t’h = few minutes) and the (S),(-) form (no aging in I8 hr). We cannot say whether the (S),( -) form of inhibited AChE ages rapidly or is refractory to oxime treatment for some other reason. The (R)p( +) form did age slightly in 18 hr at pH 8.0 or 7.0. No test was made of effect of change of pH on aging of inhibited NTE but it is known that there was little effect after inhibition by some other phosphonates (13. Predictions about comparative acute or delayed effects in vivo can often be made after studies such as are reported here of in vitro interactions of OP direct inhibitors with the target enzymes (4,9>. It is not possible to predict what dose will lead to high inhibition of AChE and acute toxic effects since distribution of the agent in the body, metabolic disposal, and excretion are dominant factors. However, the rule seems fairly vindicated that a neuropathic dose will be less than a lethal dose only if the potency of the proximal toxin against NTE is greater than that against AChE, i.e., k aNTE is greater than kaAChE. On this basis
PHENYLPHOSPHONATE
and using the ratios from Table 1 we might predict that doses of the (S),( -) isomers many times the unprotected LD,, would be required to cause high in viwo inhibition of NTE with consequent initiation of neuropathy while for the (R)r( +) isomers doses only 2-3 x LD,, given to hens with concomitant prophylaxis and therapy against acute toxicity should be sufficient to cause high inhibition of NTE. However, such prediction of responses in vivo are totally dependent on the assumption that the same chemicals are being studied in vitro and in vim.
This study has revealed that the ethyl phenylphosphonylated NTE obtained from (R)r( +)-DCB or MCB does not age significantly in vitro. The question arises as to what would happen if 100% of NTE were converted to the same steric form in vivo: would there be a neuropathic response or would birds become protected as they do when the NTE is inhibited to a non-ageable phosphinylated form (4, 5)? Delayed neuropathy and NTE studies have been performed with three pesticides with different leaving groups but which would each be expected to give ethyl phenylphosphonylated NTE. Single doses of racemic EPN, ethyl leptophos, or cyanofenphos each cause delayed neuropathy associated with high inhibition of NTE (12, 14, 20-22). However, for the latter two compounds the measured level of NTE inhibition required to initiate neuropathy seemed to be 90-100% rather than the usual level of 70-80% as found for many compounds (7). Some of these authors (20-22) suggested that, for these pesticides, some of the inhibited NTE might be in a slow-aging configuration and the present study lends credibility to that suggestion. Some work has been done in vivo with resolved isomers of EPN but not, unfortunately, with EPNO. A single dose of L( - ) EPN can cause neuropathy (12) and high inhibition of NTE was found, as expected, l-2 days after dosing (14). For D( +)-EPN single doses up to 89 mg/kg did not cause neuropathy (12) and in one bird
ISOMERS
AND NTE
141
the NTE inhibition after 50 mglkg was only 50% which is well below the threshold for initiation (14). No aging measurements were made in these studies. Subchronic feeding of hens by either stereoisomer of EPN initiated neuropathy when the dose x time was extended sufficiently (13). To clarify interpretation of the molecular events of initiation by these compounds it would be necessary to know (i) the degree of aging (if any) and the clinical response to a highly inhibitory single dose of D( +)EPN, (ii) what were the accumulated masses of inhibited NTE under subchronic regimes which enabled both isomers to cause equal clinical effect, (iii) to what extent aging had actually occurred and (iv) whether conversion of D( + )-EPN to EPNO was totally stereospecific or whether EPN underwent significant racemisation in rive in the same way as has been shown for fonofos isomers (23). Studies in viva with the easily synthesized direct-acting stereoisomers of MCB and DCB may prove very useful in a closer examination of the intimate details of the neuropathy initiation process. REFERENCES 1. M. K. Johnson, A phosphorylation site in brain and the delayed neurotoxic effect of some organophosphorus compounds. Biochem. J. 111, 487 (1969a). 2. M. K. Johnson, The delayed neurotoxic effect of some organophosphorus compounds. Identification of the phosphorylation site as an esterase, Biachem. J. 114, 711 (1969b). 3. M. K. Johnson, Initiation of organophosphate neurotoxicity, Toxicol. Appl. Pharmacol. 61, 480 (1981). 4. M. K. Johnson, The target for initiation of delayed neurotoxicity by organophosphorus esters: biochemical studies and toxicological applications. in “Rev. Biochem. Toxicol.” (E. Hodgson, J, R. Bend, and R. M. Philpot, Eds. ). p. 141 Elsevier, New York (1982). 5. M. K. Johnson, The primary biochemical lesion leading to the delayed neurotoxic effects of some organophosphorus esters, J. Neurochem. 23, 785 (1974). 6. B. Clothier and M. K. Johnson, Rapid aging of neurotoxic esterase after inhibition by diiso-
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propyl phosphorofluoridate, Biochem. J. 177, 549 (1979). M. K. Johnson, Organophosphorus esters causing delayed neurotoxic effects: Mechanism of action and structure/activity studies, Arch. Toxicol. 34, 259 (1975a). M. K. Johnson, Structure-activity relationships for substrates and inhibitors of hen brain neurotoxic esterase. Biochem. Pharmacol. 24, 797 (1975c). M. Lotti and M. K. Johnson, Neurotoxicity of organophosphorus pesticides: Predictions can be based on in vitro studies with hen and human enzymes, Arch. Toxicol. 41, 215 (1978). H. P Benschop, C. A. G. Konings, J. Van Genderen, and L. P. A. De Jong. Isolation, anticholinesterase properties and acute toxicity of the four stereoisomers of the nerve agent soman, Toxicol. Appl. Pharmacol. 72, 61 (1984). M. K. Johnson, D. J. Read, and H. P. Benschop, Interaction of the four stereoisomers of soman with acetylcholinesterase and neuropathy target esterase of hen brain, Biochem. Pharmacol., 34, 1945 (1985). H. Ohkawa, N. Mikami, Y. Okuno, and J, Miyamoto, Stereospeciticity in toxicity of the optical isomers of EPN, Bull. Environ. Contam. Toxicol. 18, 534 (1977). M. B. Abou-Donia, D. G. Graham, A. A. Komeil, A. A. Nomeir, and W. C. Dauterman, Stereospecificity of cholinergic and delayed neurotoxic effects of O-ethyl 0-Cnitrophenyl phenylphosphonothioate optical isomers: Effect of daily oral administration to hens, in “Advances in Neurotoxicology” (L. Manzo, Ed.), p. 237 Pergamon, Oxford (1980). H. Ohkawa, H. Oshita, and J. Miyamato, Comparison of inhibitory activity of various organophosphorus compounds against acetylcholinesterase and neurotoxic esterase of hens with
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respect to delayed neurotoxicity, Biochem. Pharmacol. 29, 2721 (1980). B. Clothier and M. K. Johnson, Reactivation and aging of neurotoxic esterase inhibited by a variety of organophosphorus esters, Biochem. J. 185, 739 (1980). H. Yoshikawa, T. Shono, and M. Eto, A facile synthesis and insecticidal activity of optically active phenylphosphonothiolates, J. Pestic. Sci. 9, 455-462 (1984). B. Clothier, M. K. Johnson, and E. Reiner, Interaction of some trialkylphosphorothiolates with acetylcholinesterase: Characterisation of inhibition, aging and reactivation, Biochim. Biophys. Acta. 660, 306 (1981). B. Clothier, “Reactivation and aging of esterases,” M. Phil. Thesis. Council for Nat. Academ. Awards, London (1979). P. W. Lee, R. Allahyari, and T. R. Fukuto, Studies on the chiral isomers of Fonofos and Fonofos oxon. 1. Toxicity and antiesterase activity. Pestic. Biochem. Physiol. 8, 146 (1978). H. H. Reinders, L. G. Hansen, R. L. Metcalf, and R. A. Metcalf. In vitro and in vivo inhibition of chicken brain neurotoxic esterase by leptophos analogs, Pestic. Biochem. Physiol. 28, 67 (1983). L. G. Hansen, E. M. Cozzi, R. L. Metcalf, and T. K. Hansen, Effect of disposition on the relative delayed neurotoxicity potency among leptophos analogues. Pestic. Biochem. Physiol.. in press. N. S. Ahmed, K. S. El-Gendy, J. D. Farmer, R. Novak, N. Bakry, and S. A. Soliman, Timecourse and dose-dependent in vivo effect of ethyl-leptophos on hen brain neurotoxic esterase activity, Toxicol. Let?. 18 (Suppl. I), 111 (1983). P. W. Lee, R. Allahyari, and T. R. Fukuto, Studies on the chiral isomers of Fonofos and Fonofos oxon. II. In vitro metabolism, Pestic. Biochem. Physiol. 8, 158 (1978).