Chem. Res. Toxicol. 1997, 10, 1045-1048
1045
Phenyl Valerate Esterases Other than Neuropathy Target Esterase and the Promotion of Organophosphate Polyneuropathy† Dejan Milatovic,‡ Angelo Moretto, Khaled A. Osman,§ and Marcello Lotti* Istituto di Medicina del Lavoro, Universita` degli Studi di Padova, Via Facciolati, 71, I-35127 Padova, Italy Received December 17, 1996X
Certain esterase inhibitors (such as phenylmethanesulfonyl fluoride, PMSF) enhance the clinical and morphological signs of organophosphate-induced delayed polyneuropathy (OPIDP) in hens. This is called promotion of OPIDP. The target of promotion is unknown, but it is likely to be different from neuropathy target esterase (NTE), the target of OPIDP. NTE is a neural phenyl valerate (PV) esterase, operationally defined by selective inhibition with organophosphates. This study was aimed to ascertain whether the target for promotion is a PV esterase other than NTE. Brain and sciatic nerve PV esterases of hens were incubated with diisopropylphosphorofluoridate (DFP; 5 µM) or N,N-diisopropyl phosphorodiamidofluoridate (mipafox; 50 µM) to inhibit NTE and other esterases thought not to be relevant to promotion. Remaining activities, quantitatively similar after either inhibition, were titrated with PMSF (up to 500 µM) and analysis of time course of inhibition showed first-order kinetics. Mipafox (50 µM)-resistant PMSF (500 µM)-sensitive activity (about 80% of mipafox-resistant ones) was tested both in vitro and in vivo with several inhibitors. No correlation was found between inhibition of mipafox-resistant PMSF-sensitive activity and the capability of several inhibitors to promote OPIDP. We conclude that the target of promotion is unlikely to be a PV esterase resistant to mipafox (50 µM).
Introduction Organophosphate-induced delayed polyneuropathy (OPIDP)1 is an axonal degeneration of peripheral nerves and spinal cord caused by certain organophosphorus compounds (OP). Several cases of OPIDP have been reported in humans (1). The target of OPIDP is thought to be neuropathy target esterase (NTE), a neural phenyl valerate (PV) esterase defined as the activity resistant to the non-neuropathic paraoxon (diethyl p-nitrophenyl phosphate; 40 µM) and sensitive to the neuropathic mipafox (N,N-diisopropyl phosphorodiamidofluoridate; 50 µM). High NTE inhibition (>70%) caused by neuropathic OPs and measured soon after dosing correlates with the development of OPIDP 2 weeks later (1-4). PV esterases may be classified as subsets of the carboxylic ester hydrolases (EC 3.1.1.1). Certain esterase inhibitors, such as phenylmethanesulfonyl fluoride (PMSF), exacerbate the clinical and morphological signs of OPIDP when given after neuropathic OPs, and the phenomenon is called promotion of OPIDP (5, 6). Cases of promotion of OPIDP in humans have not been reported, nor has the phenomenon been † Part of this work was presented at the International Congress of Toxicology - VII, Seattle, WA, July 2-6, 1995. * Correspondence should be addressed to this author. Tel: +39-49750155. Fax: +39-49-8216644. E-mail:
[email protected]. ‡ Present address: Agriculture Institute, 81000 Podgorica, Montenegro, Yugoslavia. § Present address: Pesticides Chemistry Dept., Faculty of Agriculture, Alexandria University, Chatby, Alexandria, Egypt. X Abstract published in Advance ACS Abstracts, August 15, 1997. 1 Abbreviations: BuSF, n-butanesulfonyl fluoride; DFP, diisopropyl phosphorofluoridate; NTE, neuropathy target esterase; OP, organophosphate; OPIDP, organophosphate-induced delayed polyneuropathy; PMSF, phenylmethanesulfonyl fluoride; PV, phenyl valerate; p-TSF, p-toluenesulfonyl fluoride; MRPS activity, mipafox (50 µM)-resistant PMSF (500 µM)-sensitive phenyl valerate activity.
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observed out of the laboratory. However, promotion appears to be an interesting laboratory model to investigate neurological disorders. The target for promotion is unknown, and it is assumed to be a serine hydrolase; promoters identified so far are all NTE inhibitors (510), but indirect evidence suggests that it is unlikely to be NTE because the phosphorothioic acid O-(2-chloro2,3,3-trifluorocyclobutyl) O-ethyl S-propyl ester exacerbates OPIDP when given at a dose below that which would inhibit NTE (8). Initiation of OPIDP by NTE phosphorylation and promotion of OPIDP by inhibition of an unknown esteratic site both occur in axons and not in the cell body (11-13). For these reasons it has been suggested that the promotion target should be similar to and/or “linked” with NTE (14). Studies reported here were performed to ascertain whether the target of OPIDP promotion is a PV esterase other than NTE. Brain and sciatic nerve total PV esterases were inhibited with either mipafox or, less selectively, diisopropyl fluorophosphate (DFP) to remove NTE. The remaining PV esterase activity was then titrated with the promoter PMSF, and most of it was inhibited with first-order kinetics. This PV activity was considered as the possible site for promotion, and its sensitivity to promoters and nonpromoters was studied both in vitro and in vivo.
Materials and Methods Caution: The following chemicals are hazardous and should be handled carefully: BuSF, DFP, L-(-)- and D-(+)-methamidophos, mipafox, paraoxon, and PMSF. Use gloves when handling the pure compound or concentrated solutions. Strong alkaline solutions (NaOH) should be used for rapid degradation of the compounds. Chemicals. Mipafox (purity >98%) and PV were purchased from Lark Enterprises (Webster, MA); paraoxon was purchased
© 1997 American Chemical Society
1046 Chem. Res. Toxicol., Vol. 10, No. 9, 1997 from Sigma Chemical Co. (St. Louis, MO) and purified according to Johnson (4); PMSF (purity >99%) and DFP (purity >95%) were purchased from Fluka AG (Buchs, Switzerland); p-toluenesulfonyl fluoride (p-TSF; pure) was purchased from Aldrich Chemie (Steinheim, Germany); L-(-)- and D-(+)-methamidophos (O,S-dimethyl phosphorothioamidate; pure) were a gift of Bayer AG (Leverkusen, Germany); n-butanesulfonyl fluoride (BuSF; pure) was purchased from Oryza Labs (Newburyport, MA). All compounds were dissolved in acetone (1% final concentration), except mipafox and methamidophos isomers which were dissolved in Tris (pH 8.0) when used in vitro. All compounds were dissolved in glycerol formal (purchased from Fluka AG, Buchs, Switzerland) immediately before use for in vivo studies, except methamidophos isomers which were dissolved in saline. Animals and Tissues. Randomly bred adult hens (1.7-2.5 kg of body weight) were purchased from a local breeder and allowed food and water ad libitum. Animals were killed by decapitation. Brain and sciatic nerves were excised and either assayed immediately or stored at -80 °C prior to assay. Titrations of PV Esterases Other than NTE. Tissues from untreated hens were homogenized (brain, 100 mg‚mL-1; sciatic nerve, 40 mg‚mL-1) in 50 mM Tris (pH 8.0), containing EDTA (0.2 mM). Diluted homogenates were incubated with either mipafox (50 µM) or DFP (5 µM corresponding to about 10 times the NTE IC50), either alone or in combination with PMSF (1-500 µM) for 20 min at 37 °C prior to addition of PV. We chose the same PV concentration used for the standard NTE assay (1.3 mM) (4). PV is an artificial substrate since no endogenous substrate is known for these esterases; it is poorly soluble, and the concentration is above the limit of solubility and lower than the Km (10 mM). Hydrolysis was terminated after 15-35 min as described for the NTE assay (see below). Homogenates were diluted in order to obtain adequate absorbances at 510 nm (0.5-1.5). Appropriate blanks as controls for spontaneous hydrolysis of PV were always included (also in the assays described below). Data obtained were fitted to the following function:
%A ) (IC50 + β[I]/(IC50 + [I]) where %A is the percent activity at a given PMSF concentration ([I]), β is the residual %A at high [I], and IC50 is the concentration causing 50% inhibition. The confidence limits (p < 0.05) of the percentage of activity resistant to high PMSF concentration are reported (see Figure 1). Standard Assay of Mipafox-Resistant PMSF-Sensitive PV Esterase (MRPS Activity). Paired samples of homogenates (2.5 and 6 mg‚mL-1, w/v, for brain and sciatic nerve, respectively) were incubated with mipafox (50 µM) plus either acetone or PMSF (500 µM) for 20 min. The remainder of the assay was carried out as described for NTE. Activity was calculated from the differential absorbance at 510 nm between homogenates incubated with mipafox and those incubated with mipafox plus PMSF. Assay of NTE Activity. Brain and sciatic nerve NTE were assayed according to Johnson (4) and Moretto et al. (15), respectively. Determination of IC50s for NTE and MRPS Activity. Inhibitors were incubated for 20 min at 37 °C with homogenates, and then PV was added. Reported IC50 values were derived by regression analysis of the semilog plots of percent remaining activity vs inhibitor concentration (6-10 concentrations up to 5 × IC50, except where noted). Animal Treatment. L-(-)- and D-(+)-methamidophos were administered by gavage (1 mL‚kg-1) to hens fasted overnight. Other compounds were administered either sc (0.2-1.0 mL‚kg-1 on one or two sites of the anterothoracic region) or iv in the wing vein (0.25 mL‚kg-1). Control animals were treated with the vehicle. To counteract cholinergic toxicity, pretreatment with atropine (20 mg‚kg-1 ip) was given to animals before dosing with methamidophos isomers, paraoxon, or DFP. Animals treated with methamidophos isomers were given atropine several times after dosing, as needed. Promotion of DFP
Milatovic et al. neuropathy by BuSF was tested as follows: hens were given DFP plus, 24 h later, either vehicle or BuSF and then observed for gait disturbances up to 21 days after treatment. Ataxia was evaluated on a 0-8 point scale (6).
Results and Discussion PV esterase activity (mean ( SD) resistant to mipafox (50 µM) was 6.7 ( 0.9 (n ) 10) and 1.4 ( 0.2 (n ) 8) µmol‚min-1‚g of tissue-1 in brain and sciatic nerves, respectively. About 80% of that was sensitive to PMSF, and time course experiments (with 10 and 50 µM PMSF, data not shown) suggested that inhibition was first order. The calculated IC50s in brain and sciatic nerves were similar (Table 1). In other experiments, DFP (5 µM) was used to inhibit NTE. Remaining PV esterase activity was 6.1 ( 0.8 (n ) 10) and 1.3 ( 0.2 (n ) 4) µmol‚min-1‚g of tissue-1 in brain and peripheral nerves, respectively. Differences with the activities resistant to mipafox, although not statistically significant, suggest inhibition of a minor fraction of mipafox-resistant esterases by DFP (16). Kinetics of inhibition of DFP-resistant PV esterases by PMSF was similar to that of mipafox-resistant ones (Figure 1). It was concluded that probably a single PV esterase activity resistant to either mipafox or DFP was inhibited by the promoter PMSF, which was then considered as a candidate target for promotion. Following experiments were carried out by using mipafox to inhibit NTE. Table 1 shows the in vitro inhibition of mipafoxresistant PMSF-sensitive (MRPS) activity in brain and sciatic nerve caused by either promoters or nonpromoters. For comparison, NTE inhibition was also measured. Sensitivity to inhibitors of both MRPS and NTE activities was similar in brain and sciatic nerve. PMSF inhibited MRPS activity at concentrations slightly lower than those which inhibited NTE. BuSF displayed a comparable behavior. Table 1 also reports (see footnote a) that BuSF was a promoter of DFP neuropathy. Moreover, when BuSF was used in vitro to inhibit MRPS activity, a portion of it (about 5% and 10% in brain and sciatic nerve, respectively) was resistant to high concentrations of BuSF suggesting that MRPS activity is not a single enzyme, as was concluded on the basis of titrations with PMSF. Both isomers of methamidophos were equipotent promoters of OPIDP (9). However, the L-(-)-isomer inhibited MRPS activity at about the same concentrations needed to inhibit NTE, whereas the D-(+)-isomer was not an inhibitor of MRPS activity. DFP, which is not a promoter (17), was a much weaker inhibitor of MRPS activity than of NTE, the IC50s differing by 2 orders of magnitude. The nonpromoter p-TSF was a more potent inhibitor of MRPS activity than of NTE. Moreover, further confirmation that MRPS activity is not a single enzyme was the finding that about 8% of it in brain and 20% in sciatic nerve were resistant to high concentrations of p-TSF. These results indicated that there is no correlation between in vitro inhibition of MRPS activity and the in vivo promotion effects of these compounds. Table 2 shows the in vivo results which confirm in vitro data. The promoter PMSF, when given at the minimum promoting dose (5 mg‚kg-1 sc) (6), caused 60-70% inhibition of MRPS activity and a lower (35-40%) inhibition of NTE, reflecting the differences in IC50s. At maximum promoting dose (125 mg‚kg-1 sc), both enzymes were almost completely inhibited. BuSF also inhibited
Esterases and Organophosphate Neuropathy Promotion
Chem. Res. Toxicol., Vol. 10, No. 9, 1997 1047
Figure 1. Titration with PMSF of hen brain and sciatic nerve PV esterases resistant to 50 µM mipafox or 5 µM DFP. Data of each graph are from three different hens. Activity resistant to high PMSF concentrations (confidence limits at p < 0.05), when mipafox was used, was 4-8% and 13-15% in brain and peripheral nerve, respectively; when DFP was used, it was 11-19% and 19-26% in the same tissues, respectively. Table 1. Inhibition of MRPS and NTE Activities by Promoters and Nonpromoters in Vitro IC50 (mM) MRPS activity compounda
brain sciatic nerve
PMSF BuSF L-(-)-methamidophos D-(+)-methamidophos
Promoters 0.05 0.04 0.01c 0.01b 8d 8d NS NS
DFP p-TSF
Nonpromoters 0.05 0.04 0.02e 0.02f
NTE brain 0.1 0.06 11 0.6 0.0005 1
sciatic nerve 0.1 0.06 7 0.7 0.0005 0.8
a References for promotion/nonpromotion are PMSF (6), L-(-)and D-(+)-methamidophos (9), DFP (17), p-TSF (10). BuSF (5 mg‚kg-1 iv) promoted DFP (0.5 mg‚kg-1 sc) neuropathy. The median (range clinical score) of promoted animals was 5 (3-8), n ) 5, statistically different (p < 0.05, Mann-Whitney U-test) from that of animals treated with DFP only, which was 1 (0-3), n ) 5. b 5% of the activity was resistant to 10 × IC . c 10% of the activity 50 was resistant to 10 x IC50. d Maximum concentration tested: 20 mM. e 8% of the activity was resistant to 5 x IC50. f 20% of the activity was resistant to 5 × IC50. NS: no significant inhibition at 5 × IC50.
both enzymes when administered at promoting dose (see footnote a in Table 1), but the inhibition of MRPS activity
was lower in the sciatic nerve than in brain. Both optical isomers of methamidophos were given at 2 times the minimal promoting dose (9), but only marginal inhibition of MRPS activity was detected. Concurrently, NTE inhibition was minimal in L-(-)-methamidophos-treated and substantial in D-(+)-methamidophos-treated hens, as expected (9). DFP, given at highly neuropathic doses (17), inhibited about 90% NTE and about 40% MRPS activity. p-TSF given at doses which do not promote OPIDP (10) selectively inhibited MRPS activity, more in brain than in sciatic nerve. Paraoxon, when given at the maximum tolerated dose, does not cause promotion (6) but inhibited about 50% MRPS activity. In conclusion, MRPS activity was not inhibited by promoters such as methamidophos isomers, whereas it was inhibited to various extents by all nonpromoters. Lack of correlation may be explained by the presence of relatively small enzyme activities with different sensitivities to inhibitors which may have been overlooked by using PMSF. Indeed, dissection of enzymes by means of selective inhibition is not precise, and in fact, data with BuSF and p-TSF indicate that MRPS activity is not entirely homogeneous. Nevertheless, even considering this imprecision, the lack of correlation between inhibition of MRPS activity and the effect of promotion still remains. Therefore, the target for promotion does not
1048 Chem. Res. Toxicol., Vol. 10, No. 9, 1997
Milatovic et al.
Table 2. Inhibition of MRPS and NTE Activities by Promoters and Nonpromoters in Vivo % control activityb MRPS activity compounda mg‚kg-1, route
brain
sciatic nerve
NTE brain
sciatic nerve
Promoters PMSF 5, sc 125, sc BuSF 5, iv L-(-)-methamidophos 50, po D-(+)-methamidophos 50, po
30 ( 3 4(2
44 ( 7 8(2
66 ( 12 7(2
62 ( 11 11 ( 2
11 ( 3
37 ( 5
29 ( 7
32 ( 4
92 ( 7
87 ( 10
91 ( 7
94 ( 2
91 ( 9
91 ( 5
24 ( 7
23 ( 1
Nonpromoters DFP 2.0, sc p-TSF 240, sc paraoxon 0.6, sc
52 ( 1
66 ( 16
10 ( 1
13 ( 7
8(1
49 ( 5
101 ( 10
103 ( 7
48 ( 5
51 ( 4
90 ( 3
92 ( 7
a
See footnote a in Table 1. The doses of promoters were the effective ones. Dose of DFP was 3-4 times the minimal neuropathic one, whereas those of p-TSF and paraoxon were the maximum tolerated ones. b Measured 24 h after dosing and expressed as mean ( SD (n ) 3-5). MRPS activities of controls were (mean ( SD) 6.2 ( 0.4 (n ) 12) and 1.1 ( 0.2 (n ) 11) µmol‚min-1‚g of tissue-1 in brain and sciatic nerve, respectively. NTE activities of controls were 2.5 ( 0.3 (n ) 11) and 0.1 ( 0.01 (n ) 9) µmol‚min-1‚g of tissue-1 in the same tissues.
seem to be a PV esterase other than NTE which is defined as a PV esterase resistant to mipafox (50 µM). Until now, NTE has been defined by means of selective inhibition because its purification has only recently been achieved (18). A better characterization of NTE might help to clarify whether a minor fraction or an isoform of NTE is involved in promotion.
Acknowledgment. D. Milatovic is a recipient of an Italian Government fellowship for scientists from the Balkan Region. K. A. Osman is a recipient of a fellowship from the Italian Ministry of Foreign Affairs. We thank Dr. M. Jokanovic for performing some experiments and C. A. Drace-Valentini for preparation of the manuscript. The financial support of CNR, MURST, Regione Veneto, and Bayer AG (Germany) is gratefully acknowledged. The conclusions are those of the authors and not of the sponsors.
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(2) Johnson, M. K. (1990) Organophosphates and delayed neuropathy - Is NTE alive and well? Toxicol. Appl. Pharmacol. 102, 385399. (3) Richardson, R. J. (1992) Interactions of organophosphorus compounds with neurotoxic esterase. In Organophosphates. Chemistry, fate and effects (Chambers, J. E., and Levi, P. E., Eds.) pp 300-323, Academic Press, San Diego, CA. (4) Johnson, M. K. (1977) Improved assay of neurotoxic esterase for screening organophosphates for delayed neurotoxicity potential. Arch. Toxicol. 37, 113-115. (5) Pope, C. N., and Padilla, S. (1990) Potentiation of organophosphorus-induced delayed neurotoxicity by phenylmethylsulfonyl fluoride. J. Toxicol. Environ. Health 31, 261-273. (6) 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. (7) Johnson, M. K., and Read, D. J. (1993) Prophylaxis against and promotion of organophosphate-induced delayed neuropathy by phenyl di-n-pentyl phosphinate. Chem.-Biol. Interact. 87, 449455. (8) Moretto, A., Bertolazzi, M., and Lotti, M. (1994) The phosphorothioic acid O-(2-Chloro-2,3,3-trifluorocyclobutyl) O-ethyl Spropyl ester promotes organophosphate polyneuropathy without inhibition of neuropathy target esterase. Toxicol. Appl. Pharmacol. 129, 133-137. (9) 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. (10) Osman, K. A., Moretto, A., and Lotti, M. (1996) Sulfonylfluorides and the promotion of diisopropylfluorophosphate neuropathy. Fundam. Appl. Toxicol. 33, 294-297. (11) Lotti, M., Caroldi, S., Moretto, A., Johnson, M. K., Fish, C. J., Gopinath, C., and Roberts, N. L. (1987) Central-peripheral delayed neuropathy caused by diisopropyl phosphorofluoridate (DFP): segregation of peripheral nerve and spinal cord effects using biochemical, clinical and morphological criteria. Toxicol. Appl. Pharmacol. 88, 87-96. (12) Carrera, V., Barril, J., Mauricia, M. C., Pellin, M. C., and Vilanova, E. (1992) Local application of neuropathic organophosphorus compounds to hen sciatic nerve: inhibition of Neuropathy Target Esterase and peripheral neurological impairments. Arch. Toxicol. 88, 87-96. (13) Peraica, M., Moretto, A., and Lotti, M. (1995) Selective promotion by phenylmethanesulfonyl fluoride of peripheral and spinal cord neuropathies initiated by diisopropyl phosphorofluoridate in the hen. Toxicol. Lett. 80, 115-121. (14) Aldridge, W. N. (1993) Postscript to the symposium on organophosphorus compound induced delayed neuropathy. Chem.-Biol. Interact. 87, 463-466. (15) Moretto, A., Lotti, M., and Spencer, P. S. (1989) In vivo and in vitro regional differential sensitivity of neuropathy target esterase to di-n-butyl-2,2 dichlorovinyl phosphate. Arch. Toxicol. 63, 469473. (16) Johnson, M. K. (1988) Sensitivity and selectivity of compounds interacting with neuropathy target esterase. Further structureactivity studies. Biochem. Pharmacol. 37, 4095-4104. (17) Pope, C., Tanaka, D., Jr., and Padilla, S. (1993) The role of neurotoxic esterase (NTE) in the prevention and potentiation of organophosphorus induced delayed neurotoxicity (OPIDN). Chem.Biol. Interact. 87, 395-406. (18) Glynn, P. I., Read, D. J., Guo, R., Wylie, S., and Johnson, M. K. (1994) Synthesis and characterisation of a biotinylated organophosphorus ester for detection and affinity purification of a brain serine esterase: neuropathy target esterase. Biochem. J. 301, 551-556.
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