Organophosphorus-induced delayed neuropathy: A simple and efficient therapeutic strategy

Toxicology Letters 192 (2010) 238–244

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Organophosphorus-induced delayed neuropathy: A simple and efficient therapeutic strategy Guilherme L. Emerick a , Rosangela G. Peccinini b , Georgino H. de Oliveira b,∗ a b

Pharmaceutical Sciences – Graduation Program, School of Pharmaceutical Sciences, UNESP-São Paulo State University, Brazil Department of Natural Active Principles and Toxicology, School of Pharmaceutical Sciences, UNESP-São Paulo State University, Brazil

a r t i c l e

i n f o

Article history: Received 3 September 2009 Received in revised form 27 October 2009 Accepted 28 October 2009 Available online 13 November 2009 Keywords: TOCP Delayed neuropathy CANP Nimodipine Calcium gluconate Treatment

a b s t r a c t Organophosphorus (OP) used as pesticides and hydraulic fluids can produce acute poisoning known as OPinduced delayed neuropathy (OPIDN), whose effects take long time to recover. Thus a secure therapeutic strategy to prevent the most serious effects of this poisoning would be welcome. In this study, tri-ocresyl phosphate (TOCP, 500 mg/kg p.o.) was given to hens, followed or not by nimodipine (1 mg/kg i.m.) and calcium gluconate (Ca-glu 5 mg/kg i.v.). Six hours after TOCP intoxication, neuropathy target esterase (NTE) activity inhibition was observed, peaking after 24 h exceeding 80% inhibition. A fall in the plasmatic calcium levels was noted 12 h after TOCP was given and, in the sciatic nerve, Ca2+ fell 56.4% 24 h later; at the same time calcium activated neutral protease (CANP) activity increased 308.7%, an effect that lasted 14 days. Any bird that received therapeutic treatment after TOCP intoxication presented significant signs of OPIDN. These results suggest that NTE may be implicated in the regulation of calcium entrance into cells being responsible for the maintenance of normal function of calcium channels, and that increasing CANP activity is responsible to triggering OPIDN. Thus, with one suitably adjusted dose of nimodipine as well as Ca-glu, we believe that this treatment strategy may be used in humans with acute poisoning by neuropathic OP. © 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction The widespread use of pesticides in agriculture in order to increase crop production, has been generating a series of toxicological problems, sometimes misdiagnosed (Vasconcellos et al., 2002; Araújo et al., 2007). The World Health Organization (WHO), reports about three million cases per year of acute poisoning by pesticides, resulting in 220 thousand deaths, of which 70% occur in developing countries. A survey by the Pan American Health Organization, in 12 countries of the Latin American and Caribbean, showed that chemical poisoning, especially by lead and pesticides, represents 15% of all occupational diseases reported (WHO, 1990). A number of organophosphorus OP compounds are used as pesticides because of their ability to inhibit acetylcholinesterase in insects. Although the compounds used as insecticides show

Abbreviations: TOCP, tri-o-cresyl phosphate; OP, organophosphorus; OPIDN, organophosphorus-induced delayed neuropathy; LNTE, lymphocyte neuropathy target esterase; NTE, neuropathy target esterase; BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; PBS, phosphate buffered saline; CANP, calcium activated neutral protease; Ca-glu, calcium gluconate; CNS, central nervous system. ∗ Corresponding author. Present address: School of Pharmaceutical Sciences, UNESP-São Paulo State University, Rod. Araraquara-Jau, km 1 Campus Ville, 14801902 Araraquara, São Paulo, Brazil. Tel.: +55 16 3301 6986; fax: +55 16 3301 6980. E-mail address: [email protected] (G.H. de Oliveira). 0378-4274/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2009.10.032

preferential toxicity to insects, they are also toxic to human and other animals by the same mode of action. On the other hand, some OPs have been shown to produce long-term neurological damage independent of their effects on acetylcholinesterase. This damage, known as organophosphorus-induced delayed neuropathy (OPIDN), is characterized by axonal degeneration which is only slowly and incompletely reversible (Battershill et al., 2004). Indeed, cresyl and related phosphates are used as hydraulic fluids, lubricants, flame retardants and plasticizers (Marino and Placek, 1994). Despite tri-o-cresyl phosphate (TOCP) not being a pesticide, it was the first chemical known to produce OPIDN in human because of a huge poisoning accident in which ginger liquor was contaminated with TOCP in a classic case of intoxication reported in the USA in 1930s. Later, similar epidemics have been reported in Morocco, Netherlands, Yugoslavia, France, South Africa, Sri Lanka and India (Zhao et al., 2004). The mechanism of toxic action of OPIDN was described by Johnson (1982) and initially involves neuropathy target esterase (NTE) phosphorylation. NTE was originally identified by Johnson (1969) as a target of organophosphorus esters that cause delayed neuropathy in hens. It is a large polypeptide of 1327 amino acids membrane-bond esterase with a molecular weight of 155 kDa, and its physiological role has not yet been clarified (Glynn, 2006, 2007). Simple inhibition of NTE by OP is not sufficient to result in OPIDN. It is necessary to generate a negative charge on the terminal por-

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tion of the phosphate group bonded to the enzyme. This occurs as a result of a second reaction, so called “aging”. In this step, the cleavage of one bond in the R–O–P chain, with the loss of R leads to the formation of a charged mono-substituted phosphoric acid residue still attached to the protein. This reaction is called aging because it is a slow progressive process and the product is no longer responsive to nucleophilic reactivating agents such as oximes. This complex (enzyme–organophosphate) is very stable and resistant to breakage or hydrolytic reactivation (Glynn, 2000). Thus, the sequence of effects of OPIDN is seen as a progressive and symmetrical distal axonopathy, beginning 8–14 days after intoxication, which seems to proceed similarly to Wallerian degeneration. This kind of degeneration is characterized by the activation of calcium activated neutral protease (CANP) due to an excessive intake of calcium by the cell. The activation of CANP promotes digestion of the terminal portion of axons, preventing the transmission of nerve impulses to the post-synaptic cell (Anthony et al., 2001). Severe cases of intoxication are seldom seen in workers occupationally exposed to low or moderate levels of OP pesticides, although occasional cases have been alleged (Johnson et al., 2000). Several studies have been undertaken in an attempt to alleviate the signs and symptoms of OPIDN, some of them by replacement of calcium (Piao et al., 2003) and others by the administration of calcium-channel blockers; El-fawal et al. (1990) showed a protective effect of nifedipine in the muscles and peripheral nerves of hens treated with inducers of neuropathy. Wu and Leng (1997) also demonstrated the protective effect of verapamil which reduced the phosphorylation of protein in the brain of chickens given TOCP orally. Choudhary et al. (2006) observed the effect of nimodipine on the phosphorylation of proteins in the rat after the administration of dichlorvos. Muzardo et al. (2008) demonstrated that fast reversion of the imbalance in plasma-free Ca2+ concentration can exert not only some beneficial effect in recovering lymphocyte NTE (LNTE) activity, but also prevent the increase of neurotoxicity scores in chicken. However, the authors were unable to identify the disposition of Ca2+ . Thus, neither safe preventive nor suitable therapeutic measures against OPIDN are currently known (Richardson, 2005). In this context, the aim of this work was to investigate the mechanism of toxic action of neuropathic OP compounds through the imbalance plasma-free Ca2+ concentration, using TOCP as the prototype, so as to achieve a safer and more effective treatment for OPIDN.

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2. Materials and methods 2.1. Chemicals Tri-o-cresyl phosphate was purchased from Acros Organics, Pittsburg, PA, USA; nimodipine, sodium dodecyl sulfate, paraoxon 90%, bovine serum albumin, Coomassie Brilliant Blue G-250, Histopaque-1077, Tris (hydroxymethyl) aminomethane, casein, DEAE cellulose, ethylene diamine tetra-acetic acid (EDTA), ethylene glycol-bis(2-aminoethylether)-N,N,N ,N -tetra-acetic acid (EGTA), calcium chloride, phosphoric acid 85% and 2-mercaptoethanol were purchased from Sigma, St. Louis, MO, USA; mipafox and phenyl valerate from Oriza laboratories Inc., Chelmsford, MA, USA; sodium citrate and Triton X-100 from Rheidel-de Haën, Hannover, Germany; 4-aminoantipyrine, potassium ferricyanide, and dimethylformamide from Merck, Darmstadt, Germany; calcium Liquiform diagnostic kit from Labtest, Belo Horizonte, MG, Brazil; calcium gluconate 10% injectable ampoule from Hypofarma, Ribeirão das Neves, MG, Brazil; heparin 25,000 IU/5 ml from Roche, Rio de Janeiro, Brazil; Deltametrin (K-otrine® ) from, Bayer Cropscience Ltda, Rio de Janeiro, RJ, Brazil; piperazine citrate (Proverme® ) from Tortuga Agrarian zootechnical company, São Paulo, Brazil. All other chemicals were of analytical grade. 2.2. Animals Isabrown leghorn hens (70–90 weeks, weighing 1.6–2.3 kg) were obtained from the Hayashi farm cooperative of Guatapará, SP, Brazil. Before starting the experiments, the hens were treated to eliminate ecto-parasites and endo-parasites. The hens were sprayed with an aqueous solution of 0.6 ml/L K-otrine® to eliminate ecto-parasites. After 1 week of deltametrine spray, the birds received, in place of drinking water, an aqueous solution 4.6 g/L Proverme® , for 2 days, to eliminate endo-parasites. After this treatment, the hens were housed, four per cage, in a temperature and humidity-controlled room (24 ± 2 ◦ C and 55 ± 10% RH), with an automatic 12:12 light–dark photocycle lights on at 7 a.m. Purina feed and filtered tap water were provided ad libitum. All experimental procedures were conducted with the approval of the Research Ethics Committee of the School of Pharmaceutical Sciences of Araraquara, SP, Brazil in accordance with their guidelines for the care and use of laboratory animals (Resolution 23, 2007). All hens were sacrificed as follows: after cervical dislocation blood was collect through a small hole in the jugular vein and finally the hen was decapitated. 2.3. Experimental design Forty-eight hens were randomly divided in a control group (4 hens), experimental group (28 hens) and treated group (16 hens) as follows: all the TOCPs were administered by via oral after overnight fasting in the morning between 7 and 8 a.m. Control group (Ctrl.): In a group of 4 hens, the calcium levels in plasma, gastrocnemius muscle and sciatic nerve were determined. The LNTE and CANP activities in the sciatic nerve and gastrocnemius muscle were also assayed. Experimental group (Exp.): Each of the 28 hens received 500 mg/kg TOCP. Following this, the hens were sacrificed in groups of 4 at times: 6, 12, 24, 48 h and 8, 14 and 28 days. The same biochemical parameters were determined as in Ctrl. Treated group (Trt.): One group of 8 hens (A) and two groups of 4 hens (B and C) were given 500 mg/kg TOCP. Four of the first groups (Trt. A1) were sacrificed 24 h after TOCP administration and the other four (Trt. A2), 28 days after TOCP. Twelve

Table 1 Experimental design text table. Groups

Chemicals that were administered

Assessed parameters

TOCP (500 mg/kg p.o.)

Nimodipine (1 mg/kg i.m.)

Ca-glu (5 mg/kg i.v.)

Control (Ctrl.)

NA

NA

NA

Experimental (Exp.)

Time 0 h

NA

NA

Treated (Trt.) A1

Time 0 h

12 h after TOCP

30 min after nimodipine

A2

Time 0 h

12 h after TOCP

30 min after nimodipine

B

Time 0 h

18 h after TOCP

30 min after nimodipine

C

Time 0 h

24 h after TOCP

30 min after nimodipine

NA = not administered.

LNTE; Ca2+ into plasma sciatic nerve and gastrocnemius muscle; CANP into sciatic nerve and gastrocnemius muscle; clinical scores LNTE; Ca2+ into plasma sciatic nerve and gastrocnemius muscle; CANP into sciatic nerve and gastrocnemius muscle; clinical scores LNTE; Ca2+ into plasma, sciatic nerve and gastrocnemius muscle; CANP into sciatic nerve and gastrocnemius muscle LNTE; Ca2+ into plasma sciatic nerve and gastrocnemius muscle; CANP into sciatic nerve and gastrocnemius muscle; clinical scores LNTE; Ca2+ into plasma sciatic nerve and gastrocnemius muscle; CANP into sciatic nerve and gastrocnemius muscle; clinical scores LNTE; Ca2+ into plasma sciatic nerve and gastrocnemius muscle; CANP into sciatic nerve and gastrocnemius muscle; clinical scores

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hours after TOCP, Trt. A1 and A2 received 1 mg/kg of nimodipine i.m., and 30 min after nimodipine and 5 mg/kg of Ca-glu i.v. The second group (Trt. B), of 4 hens was sacrificed 28 days after TOCP administration, having received the same treatment as Trt. A2, but 18 h after TOCP. The third group (Trt. C) of 4 hens that was sacrificed 28 days after TOCP having received the same treatment as groups Trt. A2 and Trt. B, but, 24 h after TOCP. Nimodipine was dissolved in DMSO (4 mg/ml) and administered in the pectoral muscle; preliminary studies indicated that the vehicle had no adverse effect (El-fawal et al., 1990). Ca-glu was diluted to 20 mg/ml in sterilized water the experimental design is summurized on the Table 1. 2.4. Calcium analyses The blood was collected from the jugular vein in a tube with heparin. About 1 ml of the blood was centrifuged (5 min; 2000 rpm) and 20 ␮l plasma was used. Sciatic nerve and gastrocnemius muscle were dissected immediately after the hen was sacrificed and homogenized in saline (0.9% NaCl in deionized water), using ultrasonic vibra-cell at a final concentration 50 mg/ml. The free Ca2+ was assayed with the common clinical chemistry kit, Calcium Liquiform Labtest® , derived from the method described by Baginski et al. (1982). 2.5. Enzyme assays The lymphocyte was separated from blood with Histopaque® -1077 following Sigma diagnostic procedure sheet. Having isolated the lymphocytes, their proteins were determined by the Bradford (1976) method and all samples diluted in 50 mM Tris–HCl, 0.2 mM EDTA pH 8.0 buffer, so as to give 500 ␮g/ml. The LNTE activity was assayed as described by Schwab and Richardson (1986), but instead of counting cells, all samples were assayed in sonicated solution of 500 ␮g/ml protein. Preparation of tissue: Nerve and muscle were dissected, trimmed of external fat and connective tissue, weighed and homogenized. CANP was purified as described by Ballard et al. (1988), but the tissues were homogenized with 10 vol. ice-cold buffer A (20 mM Tris–HCl; 5 mM EDTA; 10 mM 2-mercaptoethanol, pH 7.5). DEAE cellulose was packed into glass columns (8 mm × 30 mm) and the homogenates were eluted with gradients of 0; 0.15; 0.2; 0.25 M (5 ml/gradient) NaCl in buffer A (1 g nerve or muscle; 1.5 g DEAE cellulose; 5 ml NaCl/gradient). Their proteins were determined as by Bradford (1976) and CANP activity was found in the fraction of 0.2 M NaCl for nerve and 0.25 M for muscle. CANP activity was determined as described by Buroker-kilgore and Wang (1993). Aliquots of 25 ␮g of protein were incubated with casein buffer (0.2 g casein; 0.605 g Tris–HCl pH 7.5; 0.078 g 2-mercaptoethanol and water so as to give 100 ml). Into two tubes, in the first one it was added EGTA (2000 ␮M in deionized H2 O), in the second CaCl2 (13,000 ␮M in deionized H2 O), so as to give a volume of 0.5 ml, where the concentrations of reagents were 2 mg/ml casein, 50 mM Tris, 10 mM 2mercaptoethanol, 100 ␮M EGTA or 650 ␮M CaCl2 . This solution was incubated for 30 min at 25 ◦ C, it was taken an aliquot of 50 ␮l and added 400 ␮l of Coomassie Brilliant Blue G-250 solution (23.5 ml of ethanol, 25.22 ml of phosphoric acid 85%,

Fig. 1. LNTE activity (% of control) after administration of 500 mg/kg TOCP p.o. n = 4, mean ± SD. *p < 0.05 compared to control according to ANOVA for multiple comparisons, followed by Tukey’s test. h = hour; d = day. Trt. A = 1 mg/kg nimodipine i.m. 12 h after TOCP and, 5 mg/kg Ca-glu i.v. 30 min later, Trt. B = 1 mg/kg nimodipine i.m. 18 h after TOCP and, 5 mg/kg Ca-glu i.v., 30 min later. Trt. C = 1 mg/kg nimodipine i.m. 24 h after TOCP and, 5 mg/kg Ca-glu i.v., 30 min later.

51.28 ml H2 O deionized water) and dilute with water to 1.6 ml. The absorbance was read at 595 nm. 2.6. Clinical observations To assess neurotoxicity development, a five-point scale was used as described by Muzardo et al. (2008): 0 means a normal bird; 1 is a slightly abnormal gait; 2 mild ataxia; 3 a severe ataxia accompanied by frequent collapse; and 4 complete incapacitation, that is, inability to move and permanent lateral decumbent. The hens were observed on days 8, 10, 12, 14, 16, 18, 20, 22, 26, and 28 after TOCP intoxication. 2.7. Statistical analyses Differences in quantitative parameters between groups were examined for statistical significance by ANOVA (Analysis of Variance), followed by Tukey’s test for multiple comparisons. These tests were run in Microsoft Office Excel 2007 for Windows. Differences in neurotoxicity scores were tested for statistical significance with the Kruskal–Wallis test, followed by Wilcoxon Mann–Whitney test for multiple comparisons. The non-parametric tests, were carried out in the BioEstat 5.0 program (Mamirauá, Brazil). The criterion of significance was p < 0.05 for all statistical analyses. All data are expressed as mean ± standard deviation.

Table 2 LNTE and sciatic nerve CANP activity, calcium concentrations at plasma, muscle and nerve, neurotoxicity scores after TOCP and treatment administrations in hens. Treatment

LNTE (␮mol/min/g protein)

CANP (absorbance/min/g protein)

Calcium

Sciatic nerve

Plasma (mg/dl)

8.0 ± 0.7

Exp. 6h 12 h 24 h 48 h 8 days 14 days 28 days

4.3 2.3 1.2 4.4 7.3 7.0 7.5

Trt. A1 24 h A2 28 days

1.3 ± 0.3* 6.9 ± 1.7

77.5 ± 14.9 71.0 ± 15.5

12.8 ± 1.4 16.1 ± 4.6

42.0 ± 21.0* 88.3 ± 36.2

291 ± 74 137 ± 17*

0 3

Trt. B 28 days

6.1 ± 1.6

56.7 ± 10.2

18.6 ± 5.5

73.8 ± 34.3

208 ± 72

2

Trt. C 28 days

6.0 ± 1.0

67.4 ± 23.9

16.1 ± 5.6

69.8 ± 22.4

216 ± 35

4

0.4 0.3* 0.3* 0.4* 0.4 0.2 0.4

– 137.1 ± 144.3 ± 150.5 ± 243.4 ± 252.8 ± 121.7 ±

23.3 23.7* 46.3* 44.2* 15.0* 20.5

14.2 ± 0.1

14.8 11.9 10.9 13.82 14.0 13.5 11.53

± ± ± ± ± ± ±

0.7 1.0* 2.0* 0.7 0.4 1.0 0.9*

92.5 ± 10.0

Nerve (␮g/g)

Ctrl.

± ± ± ± ± ± ±

81.8 ± 12.1

Clinical score (sum) Muscle (␮g/g)

103.8 127.3 67.8 36.8 44.3 15.6 113.0

± ± ± ± ± ± ±

23.9 35.0 30.8 13.4* 9.6* 5.7* 10.0

326 ± 64

290 257 184 325 135 133 104

± ± ± ± ± ± ±

29 72 54* 41 46* 34* 21*

0

0 0 0 0 4 11**

Data are mean ± SD of n = 4. *p < 0.05 compared to control using ANOVA for multiple comparisons, followed by Tukey’s test. **p < 0.05 compared to score zero using the Kruskal–Wallis for multiple comparisons followed by Wilcoxon Mann–Whitney’s test. h = hour; d = day. Ctrl. means zero time. Exp means seven groups that received just TOCP. Trt. A1 = hens received 500 mg/kg of TOCP and, 12 h after TOCP, 1 mg/kg of nimodipine and 30 min later 5 mg/kg Ca-glu. They were sacrificed 24 h after TOCP. Trt. A2 = hens received 500 mg/kg of TOCP and 12 h after TOCP, 1 mg/kg of nimodipine and 30 min later 5 mg/kg Ca-glu. They were sacrificed 28 days after TOCP. Trt. B = hens received 500 mg/kg of TOCP and, 18 h after TOCP, 1 mg/kg of nimodipine and 30 min later 5 mg/kg Ca-glu. They were sacrificed 28 days after TOCP. Trt. C = hens received 500 mg/kg of TOCP and 24 h after TOCP, 1 mg/kg of nimodipine and 30 min latter 5 mg/kg Ca-glu. They were sacrificed 28 days after TOCP. All TOCPs were given by gavage, all nimodipines were injected i.m., and all Ca-glu were injected i.v.

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Fig. 2. Plasma calcium (% of control) after administration of 500 mg/kg TOCP p.o. n = 4, mean ± SD. *p < 0.05 compared to control according to ANOVA for multiple comparisons, followed by Tukey’s test. h = hour; d = day. Trt. A = 1 mg/kg nimodipine i.m. 12 h after TOCP and, 5 mg/kg Ca-glu i.v. 30 min later, Trt. B = 1 mg/kg nimodipine i.m. 18 h after TOCP and, 5 mg/kg Ca-glu i.v., 30 min later. Trt. C = 1 mg/kg nimodipine i.m. 24 h after TOCP and, 5 mg/kg Ca-glu i.v., 30 min later.

3. Results 3.1. LNTE Six hours after TOCP was administrated, there was a significant inhibition of the LNTE activity, which reached maximum at 24 h, when it exceeded 80% inhibition (Fig. 1). Forty-eight hours after the poisoning, it was still possible to observe a significant difference from the control group. The enzyme activity fell from 8.0 to 1.2 ␮mol/min/g of protein (Table 2). By day 8 of the experiment practically there was practically no significant difference from to the control group and the LNTE activity remained unchanged until day 28. The treatment groups (A–C) followed accurately the behavior of the experimental group, with inhibition over 80% at 24 h after the TOCP, and on day 28 no significant difference from the control group (Fig. 1). 3.2. Plasma calcium The first significant drop in on the plasma calcium level was seen 12 h after TOCP administration. It remained up to 24 h after the poisoning, disappearing at 48 h and reappearing again 28 days after TOCP, as can be observed in Fig. 2. The plasma calcium level in the control group showed little variation at 14.2 ± 0.1 mg/dl (Table 2). On the other hand, hens that were given the therapeutic treatments after the TOCP suffered no significant alteration in plasma calcium concentration, compared to the control group.

241

Fig. 3. Calcium concentration and CANP activity (% of control) in sciatic nerve. n = 4, mean ± SD. *p < 0.05 compared to control according to ANOVA for multiple comparisons, followed by Tukey’s test. h = hour; d = day. Exp. 24 h = hens received just 500 mg/kg of TOCP by gavage and were sacrificed 24 h after TOCP. Exp. 28 days = hens received just 500 mg/kg of TOCP by gavage and they were sacrificed 28 days after TOCP. Trt. A1 = hens received 500 mg/kg of TOCP by gavage and, 12 h after TOCP, 1 mg/kg of nimodipine i.m. and 30 min later 5 mg/kg Ca-glu i.v. They were sacrificed 24 h after TOCP. Trt. A2 = hens received 500 mg/kg of TOCP by gavage and 12 h after TOCP, 1 mg/kg of nimodipine i.m. and 30 min later 5 mg/kg Ca-glu i.v. They were sacrificed 28 days after TOCP. Trt. B = hens received 500 mg/kg of TOCP by gavage and 18 h after TOCP, 1 mg/kg of nimodipine i.m. and 30 min later 5 mg/kg Ca-glu i.v. They were sacrificed 28 days after TOCP. Trt. C = hens received 500 mg/kg of TOCP by gavage and, 24 h after TOCP, 1 mg/kg of nimodipine i.m. and 30 min later 5 mg/kg Ca-glu i.v. They were sacrificed 28 days after TOCP.

3.4. Muscle The calcium level found in the gastrocnemius muscle of the control group hens was 92.5 ␮g/g of tissue. The fall in the muscle calcium level appeared only 48 h after TOCP administration and lasted up to day 14 (Table 2). On day 28, the muscle calcium concentration did not differ significantly from that in the control group, in contrast to plasma and sciatic nerve calcium. Among the hens that received strategic treatments, only treatment A showed a reduction in the calcium gastrocnemius muscle concentration 24 h after TOCP administration (Fig. 4). Twenty-four hours after TOCP, a reduction

3.3. Sciatic nerve After the administration of the TOCP a reduction of the calcium level appeared in the sciatic nerve, parallel to the drop seen in plasma calcium. Twenty-four hours after TOCP, the calcium concentration fell from 326 to 184 ␮g/g of tissue (Fig. 3). Except at 48 h after TOCP administration, when the calcium tissue concentration fall was not significant, it remained low at all times reduced (Table 2). At no time was the sciatic nerve calcium concentration greater than that in control group. Paradoxically, for the therapeutic strategy groups, after the poisoning, only treatment group A exhibited significant fall in the calcium concentration in the sciatic nerve (Fig. 3). Twenty-four hours after TOCP administration, CANP activity showed a significant increase, which lasted up to day 14 after TOCP administration (Table 2). The CANP activity at time zero rose from 81.8 to 252.8 absorbance/min/g of protein reaching maximum value on day 14 after TOCP administration. However, no significant difference from the control group was detected in the three therapeutic strategy groups (Fig. 3).

Fig. 4. Calcium concentration and CANP activity (% of control) in gastrocnemius muscle. n = 4, mean ± SD. *p < 0.05 compared to control using ANOVA for multiple comparisons followed by Tukey’s test. h = hour, d = day. Exp. 24 h = hens received just 500 mg/kg of TOCP by gavage and were sacrificed 24 h after TOCP. Exp. 28 days = hens received just 500 mg/kg of TOCP by gavage and they were sacrificed 28 days after TOCP. Trt. A1 = hens received 500 mg/kg of TOCP by gavage and, 12 h after TOCP, 1 mg/kg of nimodipine i.m. and 30 min later 5 mg/kg Ca-glu i.v. They were sacrificed 24 h after TOCP. Trt. A2 = hens received 500 mg/kg of TOCP by gavage and 12 h after TOCP, 1 mg/kg of nimodipine i.m. and 30 min later 5 mg/kg Ca-glu i.v. They were sacrificed 28 days after TOCP. Trt. B = hens received 500 mg/kg of TOCP by gavage and 18 h after TOCP, 1 mg/kg of nimodipine i.m. and 30 min later 5 mg/kg Ca-glu i.v. They were sacrificed 28 days after TOCP. Trt. C = hens received 500 mg/kg of TOCP by gavage and, 24 h after TOCP, 1 mg/kg of nimodipine i.m. and 30 min later 5 mg/kg Ca-glu i.v. They were sacrificed 28 days after TOCP.

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Table 3 Time course of the OPIDN scores of groups that received TOCP and treatment with nimodipine and Ca-glu. Groups

Exp Trt. A Trt. B Trt. C

Days after TOCP intoxication 12

14

2 0 0 0

4 1 0 0

16 7* 2 0 2

18 7* 2 1 3

20 8* 2 2 3

22 9* 3 2 4

26

28

10* 3 2 4

11** 3 2 4

All TOCPs were given by gavage, all nimodipines were injected i.m., and all Ca-glu were injected i.v. Exp. means seven groups that received just TOCP. Trt. A = hens received 500 mg/kg of TOCP and 12 h after TOCP, 1 mg/kg of nimodipine and 30 min later 5 mg/kg Ca-glu. They were sacrificed 28 days after TOCP. Trt. B = hens received 500 mg/kg of TOCP and 18 h after TOCP, 1 mg/kg of nimodipine and 30 min latter 5 mg/kg Ca-glu. They were sacrificed 28 days after TOCP. Trt. C = hens received 500 mg/kg of TOCP and 24 h after TOCP, 1 mg/kg of nimodipine and 30 min later 5 mg/kg Ca-glu. They were sacrificed 28 days after TOCP. Clinical scores are the sum of clinical signs of 4 hens. * p < 0.05 compared to score zero. ** p < 0.05 compared to the scores shown by the birds up to day 14 after TOCP, according to the Kruskal–Wallis for multiple comparison test followed by Wilcoxon Mann–Whitney’s test.

in the CANP activity in the gastrocnemius muscle occurred, but on day 28 these was no longer any significant difference from the control group (Fig. 4), while in no time was any significant difference in CANP activity observed in the birds that received therapeutic treatments (Fig. 4). 3.5. Clinical scores The motor activity of the hens was observed for 28 days and the neurotoxicity score for each group represents the sum of clinical signs of 4 hens. The hens of the control group had score zero. The ataxia signs started appearing on day 12 after TOCP administration. These score increased significantly on day 16 and the sum reached the maximum score 11 on day 28 as can be seen in Table 3. However, for the groups of birds that received strategic treatments after TOCP administration, no significant difference from zero time was observed throughout the 28 days of clinical observations. 4. Discussion As a target of neuropathic OP, NTE was first identified by Johnson (1969) and its inhibition is an initial event in the appearance of the delayed neuropathy in hens. It is a membrane-bound enzyme, with a molecular weight of 155 kDa. Even though it is known that this enzyme is involved in the homeostasis of the phosphatidylcholines (Vose et al., 2008), its physiological role is not yet defined (Glynn, 2007). However, for OPIDN to develop, NTE has to show inhibition and aging above 70–80% (Johnson, 1982), but later it was shown that OPIDN can develop after NTE inhibition lower than 70% (30–40%) and without aging reaction when PMSF was given afterwards (Lotti, 2002) The activity of the NTE can be measured in the central nervous system and peripheral nerves, as well as in the lymphocytes and platelets (Maroni and Bleecker, 1986). de Oliveira et al. (2002) monitoring the activity of NTE in the CNS of hens, found that the maximum inhibition of the enzyme occurred 24 h after TOCP administration. In the present study, 24 h after the TOCP was given more than 80% inhibition of the LNTE was seen in the lymphocytes (Fig. 1). All the hens of this group presented clinical signs of neuropathy 11 days after LNTE inhibition over 80%, strengthening the idea of this end point as a good biomarker for OPIDN as already observed by Lotti and Johnson (1978). Inhibitors of NTE, such as phosphinates, carbamates, and sulfonyl fluorides, which react covalently with the NTE active center, but do not promote the enzyme aging reaction, do not form a

terminal group with a negative charge, so the enzyme can be reactivated by endogenous factors. When some of these NTE inhibitors are given to birds before neuropathic OP, aging of the enzyme–OP complex does not occur rather, there is a protection of the bird, preventing the subsequent OPIDN (Moretto, 2000). However, when one of them is given after neuropathic OP, they normally exert a potentiating action, increasing the OPIDN neuropathic clinical signs (Moretto et al., 1992). A significant fall in the plasma calcium concentration has observed after TOCP administration (Piao et al., 2003). In the present study, significant reductions in the plasma calcium levels were observed 12 and 24 h and 28 days after TOCP administration, rather than a downward trend, the time course of plasma calcium shows a disrupted calcium ion balance, albeit without the Ca2+ level rising about the control (0 h) at any time (Fig. 2). Luttrell et al. (1993) also showed that after TOCP was given to hens, there was a significant fall in the calcium concentration in their sciatic nerve of hens. In the present study, after TOCP was administered the same loss of control occurred in the sciatic nerve as in the plasma (Fig. 3). This result suggests that calcium may migrate from extracellular to intracellular fluid after the poisoning. The calcium concentration in the extracellular fluid decreased while the intracellular level increased. Since CANP is an intracellular enzyme this assertion may be true, because a simultaneous rise in CANP activity occurred in sciatic nerve cells. In fact, in the present study, the sciatic nerve CANP activity corroborates this hypothesis, because while calcium in the sciatic nerve and plasma decreased and remains low up to day 14 after TOCP, the CANP activity increases and remains high until day 28 (Fig. 3). El-fawal et al. (1990) also reported that the activity of this protease in the sciatic nerve increased after TOCP was administered to hens. Therefore, after TOCP administration alterations appear in the homeostasis of calcium in diverse tissues of the organism due to increased calcium membrane permeability. This ion which is found in higher concentration outside than inside the cell, starts migrating into the cell easily, by means of altered calcium canals and activating the CANP that provokes the neurodegenerative process. According to this sequence of events, this is a typical Wallerian degeneration. According to Anthony et al. (2001), the activation of CANP promotes protein digestion in the axon terminal hindering the transmission of the nerve impulse. In the present study this sequence was clearly observed when after administration of TOCP: 6 h there was a fall in the NTE activity (Fig. 1), at 12 h there was a fall in the calcium levels in the plasma and a rise in the sciatic nerve (Fig. 2 and Table 2), and finally at 24 h, a great rise in CANP activity was seen (Fig. 3). These results suggest that the NTE may be implicated in the regulation of calcium entry into cells and the sequence of events also suggests that NTE is responsible for maintaining the normal function of calcium channels in the cellular membrane and the raised CANP activity is responsible for triggering OPIDN. The calcium level in the interstitial fluid of the muscular tissue was lower than the calcium level in the sciatic nerve; in fact the muscle needs a high intracellular level of the ion to during the muscular contraction. After TOCP is administered there was a fall in the muscle calcium level 48 h after the poisoning that lasted up to day 14 (Table 2). Despite this, on day 28 the muscle calcium concentration was not significantly different from the control group, differently from what occurred in the plasma calcium and in the sciatic nerve. However, paradoxically, the activity of the CANP at 24 h after TOCP showed a significant fall relative to the control group. Notwithstanding this paradox, these results suggest that the TOCP caused a neuropathy and not a myopathy in these hens (Fig. 4). Based on this sequence of biochemical events, a treatment strategy for OPIDN was outlined, involving a calcium canal blocker followed by replacement of the calcium in the extracellular medium, at various times after TOCP administration.

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Nimodipine is a Ca2+ channel blocker type L, belonging to the dihydropyridine class. It was synthesized to produce muscular relaxation and, as it has high lipophilicity it could be the drug of choice in view of its ability to cross the blood–brain barrier. Choudhary et al. (2006) observed the protective effect of nimodipine against the phosphorylation of cytoskeletal proteins of rats after administration of dichlorvos. Good results were obtained by Piao et al. (2003) who gave Ca-glu in the form of Calcicol® for the replacement of calcium ions in hens after TOCP and leptophos poisoning. Muzardo et al. (2008) showed that the administration of Ca-glu after TOCP poisoning prevented the loss of plasma calcium ion balance in hens. In the present study it can be observed (Fig. 1) that the administration of the nimodipine followed for Caglu had no influence on the LNTE activity either 24 h or 28 days after TOCP was given. This shows that the therapeutic strategy did not interfere with the mode of action of neuropathic OP or phosphinates and carbamates. On the other hand, the treatment did prevent the fall in plasma calcium at 24 h and 28 days, in the three times analyzed after TOCP administration (Fig. 2). Moreover, this treatment strategy blocked the excessive entry of plasma calcium into the cell thus preventing the ion imbalance in sciatic nerve calcium, so that no rise in CANP activity was observed (Fig. 3). Twenty-four hours after TOCP poisoning, the hens of the treated group A1 showed a significant fall in muscular calcium. As the CANP activity in this same group increased relative to the control group, this suggests that nimodipine does not have good availability in this tissue; this could be also result of pharmacokinetic profile of nimodipine (Fig. 4). However, 28 days after the TOCP poisoning, for all the treatments, there was neither significant change in the muscular calcium level nor in the CANP activity, suggesting slow availability of the nimodipine in this tissue. The birds were also evaluated clinically during the period of 28 days and classified in a scale of 0–4 for ataxia severity. The sum of scores of the experimental group reached 11, which was significantly different from zero. As with the biochemical parameters, only the experimental group showed a significant difference from zero. In particular the three treatment groups did not differ significantly from the score at zero time (Table 3). This result was satisfactory, because the birds did not present the most serious signs of OPIDN, even when the treatment began 24 h after TOCP administration. As 24 h after TOCP administration was the time of maximum NTE inhibition, these results suggest despite the inhibition of this enzyme either is a necessary step for the appearance of OPIDN, it is most likely that CANP activation is the step that can trigger the process of neuron degeneration. Concluding, OPIDN initiated by CNAP activation, and nimodipine blocking the entry of calcium in the cell, hinders this enzyme activation, preventing the appearance of OPIDN in hens. Thus with due adjustment of the dose of nimodipine and Ca-glu, we believe that this strategy should be explored for safety and efficacy as a possible human therapeutic treatment in acute poisoning by neuropathic OP. Conflict of interest There is any conflict of interest. Acknowledgments The authors are deeply grateful to Antônio Netto Júnior and Maria Aparecida dos Santos Francisco for their technical support. The financial support for this work was provided by Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo FAPESP Grant # 8/587619; “National Council for Scientific and Technological Development

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(CNPq)” and School of Pharmaceutical Science UNESP Araraquara, São Paulo State University, Brazil (Grant # 7/10-I PADC-FCF).

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