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Sulfonyl Fluorides and the Promotion of Diisopropyl Fluorophosphate Neuropathy
FUNDAMENTAL AND APPLIED TOXICOLOGY ARTICLE NO.
33, 294–297 (1996)
0167
SHORT COMMUNICATION Sulfonyl Fluorides and the Promotion of Diisopropyl Fluorophosphate Neuropathy Sulfonyl Fluorides and the Promotion of Diisopropyl Fluorophosphate Neuropathy. OSMAN, K. A., MORETTO, A., AND LOTTI, M. (1996). Fundam. Appl. Toxicol. 33, 294–297. Phenylmethanesulfonyl fluoride (PMSF) enhances the neuropathic response when given to hens after organophosphates causing delayed polyneuropathy. This study was undertaken to ascertain whether other sulfonyl fluorides promote diisopropyl fluorophosphate (DFP) neuropathy in hens and if they inhibit neuropathy target esterase (NTE), the target for organophosphateinduced delayed polyneuropathy. Among seven sulfonyl fluoride analogs of PMSF (alkyl-, and phenylsulfonyl fluorides), only nbutanesulfonyl fluoride was found to be an NTE inhibitor in vitro at a concentration (I50 Å 60 mM) similar to that of PMSF. nButanesulfonyl fluoride (0.2 mmolrkg01 sc to hens) caused both NTE inhibition in nervous tissues (ú80%) and promotion of neuropathy after DFP (0.003 mmolrkg01 sc) similar to those observed after the same molar dose of PMSF. These results confirm that, so far, all known promoters of organophosphate polyneuropathy are also NTE inhibitors. q 1996 Society of Toxicology
Diisopropyl fluorophosphate (DFP) and other organophosphorus esters (OPs) cause a central–peripheral axonal degeneration known as organophosphate-induced delayed polyneuropathy (OPIDP). The mechanism of OPIDP initiation is thought to involve high inhibition (ú70%) of neuropathy target esterase (NTE) in nervous tissues. Certain sulfonyl fluorides, including phenylmethanesulfonyl fluoride (PMSF), prevent OPIDP when given prior to a neuropathic OP by sulfonylating at least 40–50% NTE. Initiation of and protection from OPIDP have been explained as occurring either through a molecular rearrangement of inhibited NTE, occurring with neuropathic inhibitors only (Johnson, 1990), or through differences in neuropathic power of inhibitors, being weak for protective and strong for neuropathic inhibitors (Lotti et al., 1993). In either case, when enough NTE is sulfonylated, the subsequent phosphorylation by neuropathic OPs cannot initiate OPIDP. However, clinical and morphological signs of OPIDP are exacerbated when PMSF is given after neuropathic OPs (Pope and Padilla, 1990; Lotti et al., 1991). Phosphinates (Johnson and Read, 1993) and carbamates (Lotti et al., 1991) also exacerbate OPIDP. This effect was called promotion of
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OPIDP and was found to be unrelated to NTE inhibition (Moretto et al., 1994). Thus, PMSF may cause both protection and promotion, in either case at doses which are inhibitory to NTE. When PMSF is given before a neuropathic OP, there is protection because NTE is sulphonylated and cannot be phosphorylated. Therefore, the effect on the (unknown) promotion site becomes irrelevant because neuropathy is not initiated. When PMSF is given after the neuropathic OP, promotion occurs because the neuropathy is initiated by organophosphorylation and PMSF then affects the promotion site. The purpose of this study was to ascertain whether sulfonyl fluorides other than PMSF are capable of promoting DFP neuropathy. We also determined their ability to inhibit NTE since all known promoters are NTE inhibitors and most of them promote neuropathy at doses which inhibit NTE. Acetylcholinesterase (AChE) activity was determined in conjunction with NTE because if either or both were inhibited, the access of the compound to the nervous system would be indirectly proven. MATERIALS AND METHODS Chemicals. Methanesulfonyl fluoride (MW 98, 98% purity, compound 1 from Table 1), benzenesulfonyl fluoride (MW 160, 99% purity, compound 3), 2-nitrobenzenesulfonyl fluoride (MW 205, 99% purity, compound 5), and 4-nitrobenzenesulfonyl fluoride (MW 205, 99% purity, compound 6) were purchased from Aldrich Chemie (Steinheim, Germany). 4-Methylbenzenesulfonyl fluoride (MW 174, pure, compound 4) and 4-(2-aminoethyl)benzenesulfonyl fluoride (MW 203, ú95% purity, compound 7) were purchased from Sigma Chemical Co. (St. Louis, MO). n-Butanesulfonyl fluoride (MW 140, pure, compound 2) was purchased from Oryza Labs (Newburyport, MA). Phenylmethanesulfonyl fluoride (MW 174, ú99% purity, compound 8) and diisopropyl fluorophosphate (DFP, MW 184, purity ú95%) were purchased from Fluka AG. Chem. Fabrik (Buchs, Switzerland). Diethyl p-nitrophenyl phosphate (paraoxon) was purchased from Sigma and purified according to Johnson (1977). Acetylthiocholine iodide and 5,5*-dithiobis(2-nitrobenzoic acid) were also purchased from Sigma. Phenyl valerate and N,N *-diisopropylphosphorodiamido fluoridate (mipafox) were purchased from Lark Enterprises (Webster, MA). Biochemical assays. NTE activities were measured according to Johnson (1977) in brain and spinal cord and according to Moretto et al. (1989) in sciatic nerves. Control values of NTE in brain, spinal cord, and peripheral nerve were always within historical values of the laboratory (2000–2700, 500–700, and 70–130 nmolrmin01rg of tissue01, respectively).
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SHORT COMMUNICATION Brain AChE activity was measured according to Ellman et al. (1961) with slight modification. Control values were always within historical values of the laboratory (15–25 mmolrmin01rg of tissue01). In vitro studies. Homogenates of nervous tissue from control birds were used as the source of enzymes and diluted either with 50 mM Tris– HCl buffer (pH 8.0 at 237C) containing 0.2 mM EDTA (NTE studies) or with 100 mM phosphate buffer, pH 7.4 (AChE studies). Both assays were performed at 377C. Inhibitors dissolved in acetone were added to the medium, giving a final solvent concentration of 1%, and the enzymes were inhibited for 20 min prior to substrate addition. I50s were calculated by plotting log percentage activity remaining versus sulfonyl fluoride concentrations (at least eight concentrations in the range 0.2 – 5 1 I50 or at a maximum concentration of 1 mM). Changes in pH between 7.0 and 8.0 did not affect NTE inhibition and therefore values are comparable with those of AChE obtained at pH 7.4. In vivo studies. Adult hens of Warren strain (1.7–2.7 kg body wt) were purchased from a local breeder 1 week before experiments and caged in groups of 5 to 10 with free access to standard diet and water. Paired groups of animals were injected either with DFP and sulfonylfluorides (24 h apart) and clinically assessed for 21 days (daily) or with sulfonylfluorides only and either clinically assessed or killed 24 h later by decapitation for biochemical assays, as is commonly done in OPIDP studies. Moreover, the protocol of killing birds after 24 h was chosen because (a) sulfonyl fluorides irreversibly inhibit esterases (Aldridge and Reiner, 1972) and therefore the reappearance of NTE and AChE activity is due to resynthesis only (about 10% per day); and (b) PMSF and BuSF cause peak inhibition within 24 h (Johnson, 1970) and there is no evidence that other sulfonylfluorides might have a delayed disposition. DFP and sulfonyl fluorides were dissolved immediately before use in glycerol formal and maximal volumes for sc injections were 0.2 and 1.0 mlrkg01, respectively. Control animals received glycerol formal only. Doses of sulfonyl fluorides were chosen with reference to the spectrum of PMSF-promoting doses (minimum promoting dose Å 0.2 mmolrkg01 sc, maximum promoting dose Å 0.7 mmolrkg01 sc; see Table 2). All compounds were first tested at 0.2 mmolrkg01 sc. If they were found ineffective to promote DFP neuropathy, then doses were arbitrarily increased to those corresponding to 1.7–2 times that of PMSF which caused maximum effect. No further increase in doses was attempted because of the poor solubility of these chemicals and the volumes needed for sc injections. Compound 1 was administered at a lower dose because of cholinergic toxicity (birds were pretreated with atropine, 20 mgrkg01 ip, 10 min before dosing). We limited a higher dosing of compound 7 to a few birds because of the high cost of the experiment and of phenylethanesulfonyl fluoride and phenylpropanesulfonyl fluoride because of the small amounts synthesized. A few minutes after administration of compound 2, severe convulsions occurred, which subsided within a few minutes. These convulsions did not appear related to AChE inhibition. Maximal scores of ataxia were observed 15–20 days after dosing and assessed according to Lotti et al. (1991). Brain, lumbosacral spinal cord, and peripheral nerve were excised and either placed in ice-cold 50 mM Tris–HCl buffer (pH 8.0 at 237C) containing 0.2 mM EDTA and assayed for NTE and AChE or stored at 0807C prior to assay.
RESULTS AND DISCUSSION
Table 1 shows in vitro NTE inhibition by several sulfonyl fluorides in different nervous tissues. Slight differences in I50s among tissues were within the variability already reported for several inhibitors (Moretto et al., 1989). For reference, AChE I50s were also measured. In general, sulfonyl fluorides were poor NTE inhibitors, the most potent ones
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TABLE 1 NTE and AChE Inhibition by Sulfonyl Fluorides in Vitro
Sulfonyl fluorides:
R O
S
O F I50 (mM)
Compound R 1 2 3 4 5 6 7 8
CH30 n-C4H90 C6 H 5 0 4-CH3rC6H40 2-NO2rC6H40 4-NO2rC6H40 2-NH2r4-C2H4rC6H40 C6H5rCH20
Brain
NTE spinal cord
ú1 0.06 1 1 0.6 0.4 0.4 0.1
ú1 0.06 0.9 0.8 0.5 0.3 0.5 0.1
Peripheral AChE nerve brain ú1 0.06 1.3 0.8 0.6 0.3 0.5 0.08
0.04 0.8 0.6 0.1 0.3 0.1 0.08 ú1
being n-butanesulfonyl fluoride (compound 2) and PMSF (compound 8). Methanesulfonyl fluoride (compound 1) was a very weak NTE inhibitor, as were benzenesulfonyl fluoride (compound 3) and its analogues (compounds 4, 5, 6, and 7). Compounds 1, 4, 7, and to a lesser extent 3, 5, and 6 were relatively more potent inhibitors of AChE than of NTE. These in vitro biochemical characteristics were confirmed when compounds were given to hens. Table 2 shows that NTE was consistently inhibited in different tissues by compounds 2 and 8 only, which were in turn the only promoters of DFP neuropathy. Comparing the doses to promote neuropathy, the severity of the effects, the corresponding NTE inhibitions, and NTE I50s in nervous tissues, we concluded that n-butanesulfonyl fluoride and phenylmethanesulfonyl fluoride were equipotent promoters of DFP neuropathy. Promoting doses of compounds 2 and 8 were also protective from neuropathy when given before DFP, as already reported (Johnson, 1970). Compound 7 [4-(2-aminoethyl)benzenesulfonyl fluoride] was neither an NTE inhibitor nor a promoter of DFP neuropathy. This is somewhat in contrast with a previous limited report showing that this chemical promoted DFP neuropathy when given at lower doses (0.09 and 0.12 mmolrkg01 sc) (Randall et al., 1993). We do not have any explanation for this difference. The in vitro and in vivo differences between compounds 2 and 8 and the remaining sulfonyl fluorides confirm that promoters of OPIDP so far identified are all NTE inhibitors. Only one inhibitor [phosphorothioic acid O-(2-chloro-2,3,3trifluorocyclobutyl) O-ethyl S-propyl ester] was found to promote OPIDP without NTE inhibition although doses were not much lower than those which would inhibit NTE (Moretto et al., 1994). Moreover, the L-(0)-isomer of metha-
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TABLE 2 Biochemical Effects of Sulfonyl Fluorides and the Clinical Effects on Hens When Sulfonyl Fluorides Were Given 24 hr after DFP (0.003 mmolrkg01 sc) Percentage remaining enzyme activity after sulfonyl fluoridesb NTE
Compounda
Dose (mmolrkg01, sc)
Vehicle 1 2 3 4 5 6 7 8
0.1 0.2 1.4 1.4 1.2 1.2 0.2 0.2 0.7
Brain
94 22 91 101 104 96 85 19 10
{ { { { { { { { {
12 1 8 10 8 12 5 3 1
AChE Peripheral nerve
Spinal cord
(3) (1) (6) (4) (3) (6) (3) (3) (3)
98 13 93 105 91 106 103 21 14
{ { { { { { { { {
7 3 5 2 6 10 12 3 2
(3) (3) (6) (4) (3) (6) (3) (3) (3)
68 11 69 103 100 87 87 29 15
{ { { { { { { { {
13 8 22 7 9 11 4 1 5
(3) (3) (6) (4) (3) (6) (3) (3) (3)
Brain
15 80 55 53 72 56 83 97 91
{ { { { { { { { {
2 3 5 1 8 2 4 3 4
(3) (3) (3) (3) (3) (3) (3) (3) (3)
Ataxia scores after both DFP and sulfonyl fluoridesc Median (range)
n
1 (0–3) 0 (0–0) 6 (4–8)d,e 0 (0–1) 0.5 (0–1) 0 (0–1) 1 (1–2) 1 (1–3)f 5 (3–6)d,g 8 (6–8)d
20 5 5 7 8 7 4 4 5 6
a
See Table 1. Measured 24 hr after dosing and calculated from controls whose activities [mean { SD (n)] were as follows: NTE, 2398 { 244 (13), 644 { 124 (8) and 112 { 14 (8) nmolrmin01rg of tissue01 in brain, spinal cord, and peripheral nerve, respectively; AChE, 16.2 { 2.6 (5) mmolrmin01rg of tissue01 in brain. Data are expressed as means { SD (n). c Maximal clinical score assessed 14–21 days after last dosing on a 0–8 point scale. DFP alone causes NTE inhibitions as follows: 81 { 8, 84 { 15, and 68 { 15% in brain, spinal cord, and peripheral nerve, respectively (Lotti et al., 1991). None of the sulfonyl fluorides given alone caused neuropathy at maximum tested doses (data not shown). d Different from birds treated with DFP only (Kruskal–Wallis and post-hoc comparison tests, p õ 0.05) (Dixon, 1992). e When sulfonyl fluoride was given first and DFP followed, clinical scores were 0 (0–0) (4). f One bird given 1.2 mmolrkg01 sc 24 hr after DFP had an ataxia score of 3. NTE activity 24 hr after the same dose was 88–102% in all tissues of one animal. g When sulfonyl fluoride was given first and DFP followed, clinical scores were 0 in two birds. b
midophos (O,S-dimethylphosphorothioamidate) promoted OPIDP at doses causing marginal NTE inhibition (Lotti et al., 1995). All other promoters were effective at doses causing substantial NTE inhibition. The hypothesis was made (Aldridge, 1993) that the putative promotion site, though other than NTE, should be very similar to and/or ‘‘linked’’ with NTE. These results with sulfonyl fluorides further support this intriguing hypothesis. ACKNOWLEDGMENTS K. A. Osman was the recipient of a fellowship from the Italian Ministry of Foreign Affairs. We thank Dr. D. Milatovic for his suggestions and C. A. Drace-Valentini for the preparation of the manuscript. The research reported herein has been supported by the Bayer AG, D-5090 Leverkusen, Germany. The conclusions are those of the authors and not of the sponsor. The financial support of CNR, Ministero Italiano dell’Universita` e della Ricerca Scientifica e Tecnologica, and Regione Veneto is also gratefully acknowledged.
Aldridge, W. N., and Reiner, E. (1972). Enzyme Inhibitors as Substrates. North Holland Publ. Co., Amsterdam/London. Dixon, W. J. (1992). BMDP Statistical Software Manual. Univ. of California Press, Los Angeles. Ellman, G. L., Courtney, K. K., Andres, W., Jr., and Featherstone, R. M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88–95. Johnson, M. K. (1970). Organophosphorus and other inhibitors of brain ‘‘Neurotoxic Esterase’’ and the development of delayed neurotoxicity. Biochem. J. 120, 523–531. Johnson, M. K. (1977). Improved assay of neurotoxic esterase for screening organophosphates for delayed neurotoxicity potential. Arch. Toxicol. 37, 113–115. Johnson, M. K. (1990). Organophosphates and delayed neuropathy—Is NTE alive and well? Toxicol. Appl. Pharmacol. 102, 385–399. Johnson, M. K., and Read, D. J. (1993). Prophylaxis against and promotion of organophosphate-induced delayed neuropathy by phenyl di-n-pentylphosphinate. Chem. Biol. Interact. 87, 449–455.
REFERENCES
Lotti, M., Caroldi, S., Capodicasa, E., and Moretto, A. (1991). Promotion of organophosphate induced delayed polyneuropathy by phenylmethanesulfonyl fluoride. Toxicol. Appl. Pharmacol. 108, 234–241.
Aldridge, W. N. (1993). Postscript to the symposium on organophosphorus compound induced delayed neuropathy. Chem. Biol. Interact. 87, 463– 466.
Lotti, M., Moretto, A., Capodicasa, E., Bertolazzi, M., Peraica, M., and Scapellato, M. L. (1993). Interactions between neuropathy target esterase and its inhibitors and the development of polyneuropathy. Toxicol. Appl. Pharmacol. 122, 165–171.
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SHORT COMMUNICATION Lotti, M., Moretto, A., Bertolazzi, M., Peraica, M., and Fioroni, F. (1995). Organophosphate polyneuropathy and neuropathy target esterase: Studies with methamidophos and its resolved optical isomers. Arch. Toxicol. 69, 330–336. Moretto, A., Lotti, M., and Spencer, P. S. (1989). In vivo and in vitro regional differential sensitivity of neuropathy target esterase to di-n-butyl2,2 dichlorovinyl phosphate. Arch. Toxicol. 63, 469–473. Moretto, A., Bertolazzi, M., and Lotti, M. (1994). The phosphorothioic acid-O-(2-chloro-2,3,3-trifluorocyclobutyl)-O-ethyl S-propyl ester promotes organophosphate polyneuropathy without inhibition of neuropathy target esterase. Toxicol. Appl. Pharmacol. 129, 133–137. Pope, C. N., and Padilla, S. (1990). Potentiation of organophosphorusinduced delayed neurotoxicity by phenylmethylsulfonyl fluoride. J. Toxicol. Environ. Health 31, 261–273. Randall, J. C., Jianmongkol, S., and Richardson, R. J. (1993). Potentiation of organophosphate-induced delayed neuropathy (OPDIN): selective ef-
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fect on perching ability in hens produced by serine protease inhibitors. Toxicologist 13, 123.
KHALED A. OSMAN1 ANGELO MORETTO MARCELLO LOTTI2 Universita´ degli Studi di Padova, Istituto di Medicina del Lavoro, Via Facciolati 71, 35127 Padua, Italy Received February 7, 1996; accepted July 8, 1996
1
Present address: Pesticides Chemistry Department, Faculty of Agriculture, Alexandria University, Chatby, Alexandria, Egypt. 2 To whom correspondence should be addressed. Fax: /39 49 8216644.
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SHORT COMMUNICATION Sulfonyl Fluorides and the Promotion of Diisopropyl Fluorophosphate Neuropathy Sulfonyl Fluorides and the Promotion of Diisopropyl Fluorophosphate Neuropathy. OSMAN, K. A., MORETTO, A., AND LOTTI, M. (1996). Fundam. Appl. Toxicol. 33, 294–297. Phenylmethanesulfonyl fluoride (PMSF) enhances the neuropathic response when given to hens after organophosphates causing delayed polyneuropathy. This study was undertaken to ascertain whether other sulfonyl fluorides promote diisopropyl fluorophosphate (DFP) neuropathy in hens and if they inhibit neuropathy target esterase (NTE), the target for organophosphateinduced delayed polyneuropathy. Among seven sulfonyl fluoride analogs of PMSF (alkyl-, and phenylsulfonyl fluorides), only nbutanesulfonyl fluoride was found to be an NTE inhibitor in vitro at a concentration (I50 Å 60 mM) similar to that of PMSF. nButanesulfonyl fluoride (0.2 mmolrkg01 sc to hens) caused both NTE inhibition in nervous tissues (ú80%) and promotion of neuropathy after DFP (0.003 mmolrkg01 sc) similar to those observed after the same molar dose of PMSF. These results confirm that, so far, all known promoters of organophosphate polyneuropathy are also NTE inhibitors. q 1996 Society of Toxicology
Diisopropyl fluorophosphate (DFP) and other organophosphorus esters (OPs) cause a central–peripheral axonal degeneration known as organophosphate-induced delayed polyneuropathy (OPIDP). The mechanism of OPIDP initiation is thought to involve high inhibition (ú70%) of neuropathy target esterase (NTE) in nervous tissues. Certain sulfonyl fluorides, including phenylmethanesulfonyl fluoride (PMSF), prevent OPIDP when given prior to a neuropathic OP by sulfonylating at least 40–50% NTE. Initiation of and protection from OPIDP have been explained as occurring either through a molecular rearrangement of inhibited NTE, occurring with neuropathic inhibitors only (Johnson, 1990), or through differences in neuropathic power of inhibitors, being weak for protective and strong for neuropathic inhibitors (Lotti et al., 1993). In either case, when enough NTE is sulfonylated, the subsequent phosphorylation by neuropathic OPs cannot initiate OPIDP. However, clinical and morphological signs of OPIDP are exacerbated when PMSF is given after neuropathic OPs (Pope and Padilla, 1990; Lotti et al., 1991). Phosphinates (Johnson and Read, 1993) and carbamates (Lotti et al., 1991) also exacerbate OPIDP. This effect was called promotion of
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OPIDP and was found to be unrelated to NTE inhibition (Moretto et al., 1994). Thus, PMSF may cause both protection and promotion, in either case at doses which are inhibitory to NTE. When PMSF is given before a neuropathic OP, there is protection because NTE is sulphonylated and cannot be phosphorylated. Therefore, the effect on the (unknown) promotion site becomes irrelevant because neuropathy is not initiated. When PMSF is given after the neuropathic OP, promotion occurs because the neuropathy is initiated by organophosphorylation and PMSF then affects the promotion site. The purpose of this study was to ascertain whether sulfonyl fluorides other than PMSF are capable of promoting DFP neuropathy. We also determined their ability to inhibit NTE since all known promoters are NTE inhibitors and most of them promote neuropathy at doses which inhibit NTE. Acetylcholinesterase (AChE) activity was determined in conjunction with NTE because if either or both were inhibited, the access of the compound to the nervous system would be indirectly proven. MATERIALS AND METHODS Chemicals. Methanesulfonyl fluoride (MW 98, 98% purity, compound 1 from Table 1), benzenesulfonyl fluoride (MW 160, 99% purity, compound 3), 2-nitrobenzenesulfonyl fluoride (MW 205, 99% purity, compound 5), and 4-nitrobenzenesulfonyl fluoride (MW 205, 99% purity, compound 6) were purchased from Aldrich Chemie (Steinheim, Germany). 4-Methylbenzenesulfonyl fluoride (MW 174, pure, compound 4) and 4-(2-aminoethyl)benzenesulfonyl fluoride (MW 203, ú95% purity, compound 7) were purchased from Sigma Chemical Co. (St. Louis, MO). n-Butanesulfonyl fluoride (MW 140, pure, compound 2) was purchased from Oryza Labs (Newburyport, MA). Phenylmethanesulfonyl fluoride (MW 174, ú99% purity, compound 8) and diisopropyl fluorophosphate (DFP, MW 184, purity ú95%) were purchased from Fluka AG. Chem. Fabrik (Buchs, Switzerland). Diethyl p-nitrophenyl phosphate (paraoxon) was purchased from Sigma and purified according to Johnson (1977). Acetylthiocholine iodide and 5,5*-dithiobis(2-nitrobenzoic acid) were also purchased from Sigma. Phenyl valerate and N,N *-diisopropylphosphorodiamido fluoridate (mipafox) were purchased from Lark Enterprises (Webster, MA). Biochemical assays. NTE activities were measured according to Johnson (1977) in brain and spinal cord and according to Moretto et al. (1989) in sciatic nerves. Control values of NTE in brain, spinal cord, and peripheral nerve were always within historical values of the laboratory (2000–2700, 500–700, and 70–130 nmolrmin01rg of tissue01, respectively).
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SHORT COMMUNICATION Brain AChE activity was measured according to Ellman et al. (1961) with slight modification. Control values were always within historical values of the laboratory (15–25 mmolrmin01rg of tissue01). In vitro studies. Homogenates of nervous tissue from control birds were used as the source of enzymes and diluted either with 50 mM Tris– HCl buffer (pH 8.0 at 237C) containing 0.2 mM EDTA (NTE studies) or with 100 mM phosphate buffer, pH 7.4 (AChE studies). Both assays were performed at 377C. Inhibitors dissolved in acetone were added to the medium, giving a final solvent concentration of 1%, and the enzymes were inhibited for 20 min prior to substrate addition. I50s were calculated by plotting log percentage activity remaining versus sulfonyl fluoride concentrations (at least eight concentrations in the range 0.2 – 5 1 I50 or at a maximum concentration of 1 mM). Changes in pH between 7.0 and 8.0 did not affect NTE inhibition and therefore values are comparable with those of AChE obtained at pH 7.4. In vivo studies. Adult hens of Warren strain (1.7–2.7 kg body wt) were purchased from a local breeder 1 week before experiments and caged in groups of 5 to 10 with free access to standard diet and water. Paired groups of animals were injected either with DFP and sulfonylfluorides (24 h apart) and clinically assessed for 21 days (daily) or with sulfonylfluorides only and either clinically assessed or killed 24 h later by decapitation for biochemical assays, as is commonly done in OPIDP studies. Moreover, the protocol of killing birds after 24 h was chosen because (a) sulfonyl fluorides irreversibly inhibit esterases (Aldridge and Reiner, 1972) and therefore the reappearance of NTE and AChE activity is due to resynthesis only (about 10% per day); and (b) PMSF and BuSF cause peak inhibition within 24 h (Johnson, 1970) and there is no evidence that other sulfonylfluorides might have a delayed disposition. DFP and sulfonyl fluorides were dissolved immediately before use in glycerol formal and maximal volumes for sc injections were 0.2 and 1.0 mlrkg01, respectively. Control animals received glycerol formal only. Doses of sulfonyl fluorides were chosen with reference to the spectrum of PMSF-promoting doses (minimum promoting dose Å 0.2 mmolrkg01 sc, maximum promoting dose Å 0.7 mmolrkg01 sc; see Table 2). All compounds were first tested at 0.2 mmolrkg01 sc. If they were found ineffective to promote DFP neuropathy, then doses were arbitrarily increased to those corresponding to 1.7–2 times that of PMSF which caused maximum effect. No further increase in doses was attempted because of the poor solubility of these chemicals and the volumes needed for sc injections. Compound 1 was administered at a lower dose because of cholinergic toxicity (birds were pretreated with atropine, 20 mgrkg01 ip, 10 min before dosing). We limited a higher dosing of compound 7 to a few birds because of the high cost of the experiment and of phenylethanesulfonyl fluoride and phenylpropanesulfonyl fluoride because of the small amounts synthesized. A few minutes after administration of compound 2, severe convulsions occurred, which subsided within a few minutes. These convulsions did not appear related to AChE inhibition. Maximal scores of ataxia were observed 15–20 days after dosing and assessed according to Lotti et al. (1991). Brain, lumbosacral spinal cord, and peripheral nerve were excised and either placed in ice-cold 50 mM Tris–HCl buffer (pH 8.0 at 237C) containing 0.2 mM EDTA and assayed for NTE and AChE or stored at 0807C prior to assay.
RESULTS AND DISCUSSION
Table 1 shows in vitro NTE inhibition by several sulfonyl fluorides in different nervous tissues. Slight differences in I50s among tissues were within the variability already reported for several inhibitors (Moretto et al., 1989). For reference, AChE I50s were also measured. In general, sulfonyl fluorides were poor NTE inhibitors, the most potent ones
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TABLE 1 NTE and AChE Inhibition by Sulfonyl Fluorides in Vitro
Sulfonyl fluorides:
R O
S
O F I50 (mM)
Compound R 1 2 3 4 5 6 7 8
CH30 n-C4H90 C6 H 5 0 4-CH3rC6H40 2-NO2rC6H40 4-NO2rC6H40 2-NH2r4-C2H4rC6H40 C6H5rCH20
Brain
NTE spinal cord
ú1 0.06 1 1 0.6 0.4 0.4 0.1
ú1 0.06 0.9 0.8 0.5 0.3 0.5 0.1
Peripheral AChE nerve brain ú1 0.06 1.3 0.8 0.6 0.3 0.5 0.08
0.04 0.8 0.6 0.1 0.3 0.1 0.08 ú1
being n-butanesulfonyl fluoride (compound 2) and PMSF (compound 8). Methanesulfonyl fluoride (compound 1) was a very weak NTE inhibitor, as were benzenesulfonyl fluoride (compound 3) and its analogues (compounds 4, 5, 6, and 7). Compounds 1, 4, 7, and to a lesser extent 3, 5, and 6 were relatively more potent inhibitors of AChE than of NTE. These in vitro biochemical characteristics were confirmed when compounds were given to hens. Table 2 shows that NTE was consistently inhibited in different tissues by compounds 2 and 8 only, which were in turn the only promoters of DFP neuropathy. Comparing the doses to promote neuropathy, the severity of the effects, the corresponding NTE inhibitions, and NTE I50s in nervous tissues, we concluded that n-butanesulfonyl fluoride and phenylmethanesulfonyl fluoride were equipotent promoters of DFP neuropathy. Promoting doses of compounds 2 and 8 were also protective from neuropathy when given before DFP, as already reported (Johnson, 1970). Compound 7 [4-(2-aminoethyl)benzenesulfonyl fluoride] was neither an NTE inhibitor nor a promoter of DFP neuropathy. This is somewhat in contrast with a previous limited report showing that this chemical promoted DFP neuropathy when given at lower doses (0.09 and 0.12 mmolrkg01 sc) (Randall et al., 1993). We do not have any explanation for this difference. The in vitro and in vivo differences between compounds 2 and 8 and the remaining sulfonyl fluorides confirm that promoters of OPIDP so far identified are all NTE inhibitors. Only one inhibitor [phosphorothioic acid O-(2-chloro-2,3,3trifluorocyclobutyl) O-ethyl S-propyl ester] was found to promote OPIDP without NTE inhibition although doses were not much lower than those which would inhibit NTE (Moretto et al., 1994). Moreover, the L-(0)-isomer of metha-
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SHORT COMMUNICATION
TABLE 2 Biochemical Effects of Sulfonyl Fluorides and the Clinical Effects on Hens When Sulfonyl Fluorides Were Given 24 hr after DFP (0.003 mmolrkg01 sc) Percentage remaining enzyme activity after sulfonyl fluoridesb NTE
Compounda
Dose (mmolrkg01, sc)
Vehicle 1 2 3 4 5 6 7 8
0.1 0.2 1.4 1.4 1.2 1.2 0.2 0.2 0.7
Brain
94 22 91 101 104 96 85 19 10
{ { { { { { { { {
12 1 8 10 8 12 5 3 1
AChE Peripheral nerve
Spinal cord
(3) (1) (6) (4) (3) (6) (3) (3) (3)
98 13 93 105 91 106 103 21 14
{ { { { { { { { {
7 3 5 2 6 10 12 3 2
(3) (3) (6) (4) (3) (6) (3) (3) (3)
68 11 69 103 100 87 87 29 15
{ { { { { { { { {
13 8 22 7 9 11 4 1 5
(3) (3) (6) (4) (3) (6) (3) (3) (3)
Brain
15 80 55 53 72 56 83 97 91
{ { { { { { { { {
2 3 5 1 8 2 4 3 4
(3) (3) (3) (3) (3) (3) (3) (3) (3)
Ataxia scores after both DFP and sulfonyl fluoridesc Median (range)
n
1 (0–3) 0 (0–0) 6 (4–8)d,e 0 (0–1) 0.5 (0–1) 0 (0–1) 1 (1–2) 1 (1–3)f 5 (3–6)d,g 8 (6–8)d
20 5 5 7 8 7 4 4 5 6
a
See Table 1. Measured 24 hr after dosing and calculated from controls whose activities [mean { SD (n)] were as follows: NTE, 2398 { 244 (13), 644 { 124 (8) and 112 { 14 (8) nmolrmin01rg of tissue01 in brain, spinal cord, and peripheral nerve, respectively; AChE, 16.2 { 2.6 (5) mmolrmin01rg of tissue01 in brain. Data are expressed as means { SD (n). c Maximal clinical score assessed 14–21 days after last dosing on a 0–8 point scale. DFP alone causes NTE inhibitions as follows: 81 { 8, 84 { 15, and 68 { 15% in brain, spinal cord, and peripheral nerve, respectively (Lotti et al., 1991). None of the sulfonyl fluorides given alone caused neuropathy at maximum tested doses (data not shown). d Different from birds treated with DFP only (Kruskal–Wallis and post-hoc comparison tests, p õ 0.05) (Dixon, 1992). e When sulfonyl fluoride was given first and DFP followed, clinical scores were 0 (0–0) (4). f One bird given 1.2 mmolrkg01 sc 24 hr after DFP had an ataxia score of 3. NTE activity 24 hr after the same dose was 88–102% in all tissues of one animal. g When sulfonyl fluoride was given first and DFP followed, clinical scores were 0 in two birds. b
midophos (O,S-dimethylphosphorothioamidate) promoted OPIDP at doses causing marginal NTE inhibition (Lotti et al., 1995). All other promoters were effective at doses causing substantial NTE inhibition. The hypothesis was made (Aldridge, 1993) that the putative promotion site, though other than NTE, should be very similar to and/or ‘‘linked’’ with NTE. These results with sulfonyl fluorides further support this intriguing hypothesis. ACKNOWLEDGMENTS K. A. Osman was the recipient of a fellowship from the Italian Ministry of Foreign Affairs. We thank Dr. D. Milatovic for his suggestions and C. A. Drace-Valentini for the preparation of the manuscript. The research reported herein has been supported by the Bayer AG, D-5090 Leverkusen, Germany. The conclusions are those of the authors and not of the sponsor. The financial support of CNR, Ministero Italiano dell’Universita` e della Ricerca Scientifica e Tecnologica, and Regione Veneto is also gratefully acknowledged.
Aldridge, W. N., and Reiner, E. (1972). Enzyme Inhibitors as Substrates. North Holland Publ. Co., Amsterdam/London. Dixon, W. J. (1992). BMDP Statistical Software Manual. Univ. of California Press, Los Angeles. Ellman, G. L., Courtney, K. K., Andres, W., Jr., and Featherstone, R. M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88–95. Johnson, M. K. (1970). Organophosphorus and other inhibitors of brain ‘‘Neurotoxic Esterase’’ and the development of delayed neurotoxicity. Biochem. J. 120, 523–531. Johnson, M. K. (1977). Improved assay of neurotoxic esterase for screening organophosphates for delayed neurotoxicity potential. Arch. Toxicol. 37, 113–115. Johnson, M. K. (1990). Organophosphates and delayed neuropathy—Is NTE alive and well? Toxicol. Appl. Pharmacol. 102, 385–399. Johnson, M. K., and Read, D. J. (1993). Prophylaxis against and promotion of organophosphate-induced delayed neuropathy by phenyl di-n-pentylphosphinate. Chem. Biol. Interact. 87, 449–455.
REFERENCES
Lotti, M., Caroldi, S., Capodicasa, E., and Moretto, A. (1991). Promotion of organophosphate induced delayed polyneuropathy by phenylmethanesulfonyl fluoride. Toxicol. Appl. Pharmacol. 108, 234–241.
Aldridge, W. N. (1993). Postscript to the symposium on organophosphorus compound induced delayed neuropathy. Chem. Biol. Interact. 87, 463– 466.
Lotti, M., Moretto, A., Capodicasa, E., Bertolazzi, M., Peraica, M., and Scapellato, M. L. (1993). Interactions between neuropathy target esterase and its inhibitors and the development of polyneuropathy. Toxicol. Appl. Pharmacol. 122, 165–171.
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SHORT COMMUNICATION Lotti, M., Moretto, A., Bertolazzi, M., Peraica, M., and Fioroni, F. (1995). Organophosphate polyneuropathy and neuropathy target esterase: Studies with methamidophos and its resolved optical isomers. Arch. Toxicol. 69, 330–336. Moretto, A., Lotti, M., and Spencer, P. S. (1989). In vivo and in vitro regional differential sensitivity of neuropathy target esterase to di-n-butyl2,2 dichlorovinyl phosphate. Arch. Toxicol. 63, 469–473. Moretto, A., Bertolazzi, M., and Lotti, M. (1994). The phosphorothioic acid-O-(2-chloro-2,3,3-trifluorocyclobutyl)-O-ethyl S-propyl ester promotes organophosphate polyneuropathy without inhibition of neuropathy target esterase. Toxicol. Appl. Pharmacol. 129, 133–137. Pope, C. N., and Padilla, S. (1990). Potentiation of organophosphorusinduced delayed neurotoxicity by phenylmethylsulfonyl fluoride. J. Toxicol. Environ. Health 31, 261–273. Randall, J. C., Jianmongkol, S., and Richardson, R. J. (1993). Potentiation of organophosphate-induced delayed neuropathy (OPDIN): selective ef-
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fect on perching ability in hens produced by serine protease inhibitors. Toxicologist 13, 123.
KHALED A. OSMAN1 ANGELO MORETTO MARCELLO LOTTI2 Universita´ degli Studi di Padova, Istituto di Medicina del Lavoro, Via Facciolati 71, 35127 Padua, Italy Received February 7, 1996; accepted July 8, 1996
1
Present address: Pesticides Chemistry Department, Faculty of Agriculture, Alexandria University, Chatby, Alexandria, Egypt. 2 To whom correspondence should be addressed. Fax: /39 49 8216644.
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