Soluble phenyl valerate esterases of hen sciatic nerve and the potentiation of organophosphate induced delayed polyneuropathy

Chemico-Biological Interactions 187 (2010) 340–343

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Soluble phenyl valerate esterases of hen sciatic nerve and the potentiation of organophosphate induced delayed polyneuropathy Alberto Gambalunga, Fabiola Pasqualato, Marcello Lotti ∗ Universita’ degli Studi di Padova, Dipartimento di Medicina Ambientale e Sanita’ Pubblica, Via Giustiniani 2, 30128 Padova, Italy

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Article history: Available online 25 January 2010 Keywords: Organophosphates Axonopathy Promotion/potentiation Esterase

a b s t r a c t Contrary to some organophosphorus esters (OPs), certain esterase inhibitors including sulfonyl halides, carbamates and phosphinates do not cause axonal neuropathy, but they may exacerbate traumatic and some chemical insults to axons. This phenomenon is referred to as the promotion/potentiation of axonopathies. We report here promotion studies of the organophosphate induced delayed polyneuropathy (OPIDP). This neuropathy correlates with inhibition/aging of neuropathy target esterase, but this enzyme is not the target of promotion. Soluble phenyl valerate (PV) esterases in peak I (V0 ) of hen sciatic nerve were analysed. When these activities were inhibited in vitro by a mixture containing mipafox – an OP that causes OPIDP – paraoxon and p-toluene sulfonyl fluoride – two esterase inhibitors that do not cause either neuropathy or promotion–, then the remaining activity was sensitive to classical promoters such as phenylmethane sulfonyl fluoride (PMSF) and phenylmethyl benzyl carbamate. This PV-activity was not inhibited in sciatic nerves of hens treated with di-isopropyl phosphorofluoridate, at a dose that causes OPIDP. When these birds were further dosed with PMSF a dose–response relationship was observed between inhibition of PV-esterases, as above defined, and the severity of clinical responses. These data suggest that the target of promotion is embraced in peak I (V0 ) of soluble proteins of hen sciatic nerve. © 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Organophosphate induced delayed polyneuropathy (OPIDP) is a central–peripheral axonopathy caused by certain organophosphorus esters (OPs) in sensitive species, including humans [1,2]. The putative target is an esterase in the peripheral nerve called neuropathy target esterase (NTE): when it is inhibited by OPs and the resulting phosphorylated enzyme ages, then OPIDP develops several days later. Whereas studies with genetically modified mice indicate an essential role of NTE in foetal development [3,4], only recently some light was shed on the role of NTE in the pathophysiology of axons. Studies on two families of patients with progressive spastic paraplegia associated with distal upper and lower extremity wasting disclosed an association of the disease with mutations of NTE gene [5]. Similar clinical and pathological effects are seen in NTE-deficient mice and are associated with sustained elevation of

Abbreviations: DBDCVP, di-n-butyl-2,2-dichlorovinyl phosphate; DFP, diisopropyl phosphorofluoridate; NTE, neuropathy target esterase; OP, organophosphate; OPIDP, organophosphate induced delayed polyneuropathy; PMBC, phenylmethyl benzyl carbamate; PMSF, phenyl methane sulfonyl fluoride; p-TSF, p-toluene sulfonyl fluoride; PV, phenyl valerate. ∗ Corresponding author. Tel.: +39 049 8212548; fax: +39 049 8212550. E-mail address: [email protected] (M. Lotti). 0009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2010.01.026

phosphatidyl choline [6]. Because NTE catalyses the deacylation of phosphatidylcholine, the major membrane phospholipid [7], these results suggest a role of NTE in axonal maintenance. When certain esterase inhibitors such as sulfonyl halides, carbamates and phosphinates, that do not cause neuropathy by themselves, are given to hens – the animal of choice for OPIDP – in conjuction with OPs causing OPIDP then the resulting neuropathy is more severe. This effect was called promotion of OPIDP, although others prefer to use the word potentiation. Several lines of evidence suggest that OPIDP promotion does not involve NTE, although all promoters so far identified are NTE inhibitors [8]. The search for the target of promotion started postulating the following. Given the chemical nature of promoters the target has to be an esterase similar to NTE and therefore likely to hydrolyse the same substrate – i.e. phenyl valerate (PV) – but resistant to mipafox [9,10], and it has to be present in the soluble fraction of peripheral nerves [11,12]. Early studies on hen sciatic nerve soluble fraction separated with molecular exclusion chromatography yielded two peaks of PV-activities [13], that were further characterized [11]. In previous studies, two peaks from soluble fraction of sciatic nerve (peak I or V0 peak and peak II or lower molecular weight peak) were similarly separated. Peak II was further separated with anion exchange chromatography and studied for correlation with clinical effects. However, PV-esterases of this peak were completely inhibited ex vivo by the non-promoter p-TSF [14]. The following

A. Gambalunga et al. / Chemico-Biological Interactions 187 (2010) 340–343

step included the analysis of the same proteins by using a different substrate. Several esters were tested and phenyl benzoate was chosen because it was poorly hydrolysed by NTE, but extensively by enzyme(s) that are not sensitive to non-promoters, such as mipafox (N,N -di-isopropyl phosphorodiamidofluoridrate), and sensitive to promoters such as phenyl methane sulfonyl fluoride (PMSF). However, this attempt also failed because none of these esterases was associated with clinical effects [15]. Studies are here reported aimed at exploring the PV-esterases on peak I (V0 ) of hen peripheral nerve, when separated by molecular exclusion chromatography. The strategy was based upon the selective inhibition of these esterases by chemicals with known toxicological effects and the correlations of the activities identified in this way with clinical effects. Inhibitors included promoters such as phenylmethyl benzyl carbamate (PMBC) and PMSF, neuropathic OPs such as mipafox, di-n-butyl-2,2-dichlorovinylphosphate (DBDCVP) and di-isopropyl phosphorofluoridate (DFP), and compounds that cause neither neuropathy nor promotion such as paraoxon (O,O-diethyl-p-nitrophenylphosphate) and p-toluene sulfonyl fluoride (p-TSF). 2. Materials and methods 2.1. Chemicals Mipafox (purity > 98%), chlorpyrifos-oxon (O,O-diethylO-(3,5,6-trichloro-2-piridyl) phosphotioate) and DBDCVP (purity > 98%) were purchased from Chemsyn Science Laboratories, Lenexa, KS, USA. p-TSF, paraoxon, atropine and eserine were purchased from Sigma Chemical CO., St. Louis, MO, USA. DFP (purity > 99%), PMSF (purity > 99%) and glycerol formal were purchased from Fluka AG Chem Fabrik, Buchs, Switzerland. PMBC and PV were a gift of Dr. P. Glynn (Medical Research Council, Toxicology Unit, Leicester, UK). All other reagents were of the highest analytical grade. 2.2. Animals Adult hens were purchased from a local supplier and provided feed and water ad libitum. Birds were injected subcutaneously in the anterothoracic region with the inhibitors dissolved in glycerol formal immediately before use (0.2–0.8 ml/kg). Animals were pre-treated 10 min earlier with atropine and eserine (20 mg kg−1 and 0.1 mg kg−1 respectively, i.p.) to counteract cholinergic toxicity. When multiple doses were given each chemical was administered 24 h apart. For biochemical analyses, hens were sacrificed by cervical dislocation 24 h after the last treatment and the sciatic nerves were immediately excised and processed. The clinical evaluation of ataxia was performed daily from the seventh to the twenty-first day post-treatment on a 0–8 point scale [16]. 2.3. Tissue preparation and biochemical assays Sciatic nerves were pooled (from 2 to 3 birds of each experimental group) and were homogenized (25 mg/ml) in 0.32 M sucrose/50 mM Tris (pH 8.0) containing EDTA (0.2 mM). The soluble fraction (100.000 g for 1 h) was freeze-dried overnight with a Modulyo Freeze dryer (Edwards) and re-suspended in a half volume buffer. The solution underwent molecular exclusion chromatography by loading an HighPrep 26/60 Sephacryl S-300 GE Healthcare column (volume 320 ml; 1.51 cm × 70 cm; V0 = 95 ml; fraction volume = 5 ml) with 10 mg protein, except in experiments reported in Table 3 where 20 mg protein were used. The molecular exclusion chromatography yielded two peaks of PV-esterase activities similar to those described by Escudero et al. [13]. The peak I (or V0 peak because eluted with the front, at the dead volume of the column)

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Fig. 1. Tritation of peak I (V0 ) soluble PV-esterases with mipafox. The percentage of sensitive PV-esterases was extrapolated from the intersection with the Y-axis of the non-linear part (resistant PV-esterases). IC50 was extrapolated from the linear curve (0–10 ␮M) obtained by regression analysis after subtracting resistant PV-activities.

was pooled (fractions 19–22, protein concentration 40–70 ␮g/ml, nerves from two to three hens) and the activity was measured after incubation with PV for 60 min (37 ◦ C, pH 8.0). Inhibitors were dissolved in acetone (1% solvent final concentration) with the exception of mipafox (50 mM Tris, pH 8.0) and incubated for 20 min before adding the substrate. The reaction was stopped with SDS 4%/aminoantipyrine 0.025% (w/v) and the phenol formed from the hydrolysis of the ester was measured adding K3 Fe(CN)6 (0.8%, w/v) and the colour was read at 510 nm. Fig. 1 shows the titration of PV-esterases with mipafox, as an example of calculations of sensitive fraction and related IC50 . IC50 s were extrapolated from the linear curve obtained by regression analysis after subtracting resistant PV-activities. The percentage of PV-esterases sensitive to inhibitors was derived from the intersection with the Y-axis of the non-linear part (resistant PV-esterases). Experiments were performed in triplicate using 7–10 concentration up to 10 times the IC50 s. Proteins were measured using bovine serum albumin as standard [17]. NTE activity was determined in the whole original homogenate [18]. 3. Results The sensitivity of peak I (V0 ) PV-esterases of hen’s sciatic nerve soluble fraction to various inhibitors was preliminary assessed and results are shown in Table 1. These activities were found to be relatively resistant to the non-promoter p-TSF but sensitive (about 37%) to another non-promoter, paraoxon. A substantial part of these PV-esterases was sensitive to two promoters PMSF and PMBC. Neuropathic OPs including mipafox, DFP and chlorpyrifos-oxon, inhibited this fraction to different extent. Based on these results and taking into account the estimated IC50 s of the sensitive fraction it was decided to explore the activities Table 1 Effects of inhibitors on peak I (V0 ) soluble PV-esterases from hen sciatic nerve. Inhibitor

Sensitive fraction (percentage total activity)a

IC50 of sensitive fraction (␮M) (20 min; 37 ◦ C; pH = 8)

p-TSF Paraoxon PMSF PMBC Mipafox DFP Chlorpyrifos-oxon

7 37 55 58 13 20 76

>3 2.3 124 41 2.6 0.6 0.4

a

Total activity was: 29.7 ± 3 nmol/min/mg of protein (mean ± SE, n = 5).

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Table 2 Effects of PMSF and PMBC (promoters) and DFP and DBDCVP (neuropathic OPs) on peak I (V0 ) soluble PV-esterases resistant to mipafox (50 ␮M) and p-TSF (3 ␮M) and paraoxon (2 ␮M). Inhibitor

Sensitive fraction (percentage total activity)a

PMSF IC50 of sensitive fraction (␮M) (20 min; 37 ◦ C; pH = 8)

PMSF PMBC DFP DBDCVP

74; 82 73; 75 54 37

39; 41 30; 38 14 0.08

a Total activity (remaining after mipafox, 19.9 ± 2 nmol/min/mg of protein (mean ± SE, n = 3)).

p-TSF

and

paraoxon =

Fig. 3. Titration with PMSF of peak I (V0 ) soluble PV-esterases remaining after treatment of birds with mipafox (12 mg kg−1 ) p-TSF (240 mg kg−1 ) and paraoxon (0.6 mg kg−1 ) (subtracting resistant activities). IC50 was extrapolated from the linear curve obtained by regression analysis. Table 4 Biochemical and clinical effects of PMSF in hens pre-treated with DFP (0.5 mg kg−1 ). PMSF (mg kg−1 )

Fig. 2. Titration with PMSF of peak I (V0 ) soluble PV-esterases resistant to mipafox (50 ␮M) and p-TSF (3 ␮M) and paraoxon (2 ␮M) when the resistant fraction is subtracted. Different symbols correspond to different experiments (correlation coefficients = 0.998 and 0.994).

resistant to paraoxon (2 ␮M) and p-TSF (3 ␮M) (that would inhibit esterases that are not relevant for promotion) and to mipafox (50 ␮M) (in order to inhibit NTE). The sensitivity of the remaining PV-activity to either promoters or neuropathic inhibitors was then tested (Table 2). A substantial part of it was inhibited by PMSF and PMBC with IC50 s similar to those for NTE [19], and also by DFP and DBDCVP although the IC50 s were higher than those for NTE [20]. Consequently, it was assumed that when PMSF and PMBC are given to hens at doses causing NTE inhibition, they would inhibit this fraction as well. On the contrary, DFP and DBDCVP may not inhibit this fraction when they cause in vivo NTE inhibition. The inhibition of this fraction by PMSF is likely to follow first-order kinetics (Fig. 2), when about 5–10% of total activity resistant to this inhibitor was subtracted. We attempted to measure how much of the PV-esterase activity resistant in vitro to the mixture mipafox, p-TSF and paraoxon would remain in ex vivo tissues from hens treated with the same inhibitors (Table 3). Neuropathic doses of mipafox, promoting doses of PMSF Table 3 Remaining PV-esterase activities from hens treated with mipafox, p-TSF, and either paraoxon or PMSF and their sensitivity to PMSF. Treatment

Remaining activity (percentage of total activity)a

IC50 of sensitive fraction (␮M) (20 min; 37 ◦ C; pH = 8)

Mipafoxb + p-TSFc + paraoxond Mipafoxe + p-TSFc + PMSFf Mipafoxb + p-TSFc + PMSFf

20 30 15

41 >1000 >1000

a Total PV-esterase activity from hens treated with vehicle only was 29.7 ± 3 nmol/min/mg of protein (mean ± SE, n = 5). b 12 mg kg−1 s.c. c 240 mg kg−1 s.c. d 0.6 mg kg−1 s.c. e 2 mg kg−1 s.c. f 120 mg kg−1 s.c.

0 5 30 120 240

Remaining activity (percentage of total activity)a 100b 73 30 19 19

Clinical score (n = 5) median (range) 1 (0/1–2) 2 (1/2–2) 4 (2/3–5) 7 (6/7–7) 8 (7/8–8)

a Percentage of PV-esterase activity resistant to mipafox (50 ␮M) (3 ␮M) and paraoxon (2 ␮M) and sensitive to PMSF (500 ␮M). b Control activity (after DFP) = 22.2 ± 3 nmol/min/mg of protein (mean SE, n = 3).

and maximum tolerated doses of paraoxon and p-TSF were used. The remaining PV-activities were relatively little whereas that remaining after treatment with mipafox, p-TSF and paraoxon had an IC50 for PMSF similar to that derived from naïve tissues (Table 2 and Fig. 3). No PV-activity sensitive to PMSF was found when birds were treated with mipafox, p-TSF and PMSF. When neuropathy was initiated by DFP, then the levels of ex vivo inhibition of the above fraction caused by different doses of PMSF correlated with the severity of clinical responses (promotion) (Table 4). Inhibition of the same fraction was only marginally inhibited by DFP (<5%; control activity = 23.4 ± 2 nmole/min/mg of protein; n = 3). Preliminary data also suggest that DBDCVP neuropathy may be “self promoted”. In fact, DFP neuropathy was potentiated by DBDCVP (clinical scores raising from 1 to 5) and the fraction identified as the possible target of promotion was about 50% inhibited. No further inhibition of NTE by DBDCVP was detected in these tissues. 4. Discussion The use of selective inhibitors enabled the identification of a PV-esterase activity in peak I (V0 ) of soluble hen’s sciatic nerve that, when inhibited, correlates in a dose–response fashion with the severity of promotion of the neuropathy initiated by DFP. This PVesterase activity was inhibited with first-order kinetics, indicating that it could possibly be a single protein. Whereas experiments with DFP show that OPIDP develops with negligible inhibition of the identified PV-esterase fraction, further dosing with DBDCVP suggests that the neuropathy caused by the latter may be “self promoted”. Further studies with other esterase inhibitors will ascertain as whether the correlations here reported still hold.

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