C m G a b c a A R R A A K O M C A N C 1 a t a s a ( i b n s r n S P S 0 d Toxicology 292 (2012) 145– 150 Contents lists available at SciVerse ScienceDirect Toxicology jou rn al hom epage: www.elsev ier .com/ locate / tox ico l omparative in vitro study of the inhibition of human and hen esterases by ethamidophos enantiomers uilherme L. Emericka,∗, Georgino H. DeOliveiraa, Regina V. Oliveirab, Marion Ehrichc School of Pharmaceutical Science, Department of Natural Active Principles and Toxicology, Univ Estadual Paulista – UNESP, Araraquara, SP, Brazil Department of Chemistry, Universidade Federal de São Carlos – UFSCAR, São Carlos, SP, Brazil Department of Biomedical Science and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg, VA, USA r t i c l e i n f o rticle history: eceived 18 November 2011 eceived in revised form 5 December 2011 ccepted 8 December 2011 vailable online 17 December 2011 eywords: rganophosphate pesticides ethamidophos hiral pesticides cetylcholinesterase europathy target esterase a b s t r a c t The current Organisation for Economic Co-operation and Development (OECD) guidelines for evaluat- ing organophosphorus-induced delayed neuropathy (OPIDN) require the observation of dosed animals over several days and the sacrifice of 48 hens. Adhering to these protocols in tests with enantiomers is difficult because large quantities of the compound are needed and many animals must be utilized. Thus, developing an in vitro screening protocol to evaluate chiral organophosphorus pesticides (OPs) that can induce delayed neuropathy is important. This work aimed to evaluate, in blood and brain samples from hens, human blood, and human cell culture samples, the potential of the enantiomeric forms of methami- dophos to induce acetylcholinesterase (AChE) inhibition and/or delayed neurotoxicity. Calpain activation was also evaluated in the hen brain and SH-SY5Y human neuroblastoma cells. The ratio between the inhi- bition of neuropathy target esterase (NTE) and AChE activities by the methamidophos enantiomers was evaluated as a possible indicator of the enantiomers’ abilities to induce OPIDN. The (−)-methamidophos alpain exhibited an IC50 value approximately 6 times greater than that of the (+)-methamidophos for the lym- phocyte NTE (LNTE) of hens, and (+)-methamidophos exhibited an IC50 value approximately 7 times larger than that of the (−)-methamidophos for the hen brain AChE. The IC50 values were 7 times higher for the human erythrocyte AChE and 5 times higher for AChE in the SH-SY5Y human neuroblastoma cells. Considering the esterases inhibition and calpain results, (+)-methamidophos would be expected to have OPID a greater ability to induce . Introduction Although the organophosphorus compounds (OPs), employed s insecticides exhibit preferential toxicity to insects, they are also oxic to humans and other animals due to the inhibition of AChE nd the subsequent accumulation of acetylcholine at the neuron ynapses (Johnson et al., 2000). In addition, some OPs can inhibit nd age another esterase, known as the neuropathy target esterase NTE) (Johnson, 1988), and cause a delayed effect that is known as Abbreviations: OP, organophosphorus pesticide; OPIDN, organophosphorus- nduced delayed neuropathy; AChE, acetylcholinesterase; BChE, utyrylcholinesterase; NTE, neuropathy target esterase; LNTE, lymphocyte europathy target esterase; TOCP, tri-ortho-cresyl phosphate; CNS, central nervous ystem; IC50, inhibitory concentration of 50% of enzyme activity; ki, bimolecular ate constant of inhibition; LD50, median lethal dose; OECD, Organisation for Eco- omic Co-operation and Development; ATCC, American Type Culture Collection; D, standard deviation. ∗ Corresponding author at: School of Pharmaceutical Sciences, Univ Estadual aulista – UNESP, Rod. Araraquara-Jau km 1 Campus Ville 14801 – 902, Araraquara, ão Paulo, Brazil. Tel.: +55 16 3301 6989; fax: +55 16 3301 6980. E-mail address: glemerick@yahoo.com.br (G.L. Emerick). 300-483X/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. oi:10.1016/j.tox.2011.12.004 N than the (−)-methamidophos in humans and in hens. © 2011 Elsevier Ireland Ltd. All rights reserved. organophosphorus-induced delayed neuropathy (OPIDN). OPIDN is characterized by a central–peripheral distal axonopathy and Wallerian-type degeneration that develops 8–14 days after poi- soning by a neuropathic OP (Jortner et al., 2005). The OPs that cause OPIDN include phosphates, phosphonates and phosphorami- dates. Some examples of compounds that have been reported to cause OPIDN include tri-o-cresyl phosphate (TOCP), methami- dophos, mipafox, dichlorvos and leptophos (Johnson, 1975, 1981; Lotti, 1992). However, the simple inhibition of NTE by OPs is not sufficient to cause OPIDN, which occurs along with the acute effects observed after AChE inhibition. Generating a negative charge on the terminal portion of the phosphate group bonded to the enzyme is also nec- essary and occurs as a result of a second reaction, known as “aging.” In this step, the cleavage of one bond in the R O P chain and the loss of R lead to the formation of a charged mono-substituted phos- phoric acid residue that is still attached to the protein. The “aging” reaction is possible when the OP has its radical R attached to the central phosphorus atom through a connection P O R or P S R. This reaction is called “aging” because it is a progressive process and the product is no longer responsive to nucleophilic reactivating agents (Glynn, 2000). dx.doi.org/10.1016/j.tox.2011.12.004 http://www.sciencedirect.com/science/journal/0300483X http://www.elsevier.com/locate/toxicol mailto:glemerick@yahoo.com.br dx.doi.org/10.1016/j.tox.2011.12.004 146 G.L. Emerick et al. / Toxicolog m a a i v t a e 1 c r P i o i d 1 e ( m e h i T d n 1 b c e b a e o h 2 b r t b u i i t b G 2 2 C a a c f Fig. 1. Chemical structure of methamidophos. *Chiral center. Current OECD guidelines (OECD 418, 1995; OECD 419, 1995) andate the clinical observation of dosed animals for 21 or 48 days nd the sacrifice of 48 hens as the experimental model for evalu- ting OPIDN. Following these protocols in tests with enantiomers s difficult because to obtain large quantities of these isomers is ery exhaustive and expensive. Several in vitro methods using cul- ured neuroblastoma cells or tissue homogenates (blood and brain) re employed before the in vivo methods to avoid unnecessary xpenses and excessive animal sacrifices (Fedalei and Nardone, 983; Ehrich et al., 1997). Methamidophos (O,S-dimethyl phosphoramidothioate), which ontains an asymmetric center at the phosphorus atom and one adical attached to the central phosphorus through a connection O R and the other through a connection P S R (Fig. 1), is an nsecticide widely used in agriculture, both in developed and devel- ping countries (Lin et al., 2006). Several previous studies have nvestigated the ability of methamidophos or its analogues to cause elayed neuropathy in hens (Vilanova et al., 1987; Johnson et al., 989, 1991; Bertolazzi et al., 1991; Lotti et al., 1995). McConnell t al. (1999) provided a case report suggesting that lymphocyte NTE LNTE) inhibition would predict OPIDN in patients who ingested ethamidophos. They suggested that reference values of this sterase in lymphocytes could be used as a bioindicator of OPIDN in umans. However, the potential of the racemate methamidophos n inducing neuropathy could be greater in human than in hens. his was suggested by a study in which the racemate methami- ophos was administered to hens without the development of europathy because the cholinergic crisis was so severe (Lotti et al., 995). One possible explanation for the differential effects observed etween humans and hens is the fact that this compound has a hiral center in its chemical structure and, thus, the compound xists as two enantiomers. When the racemic mixture reaches the loodstream, the enantiomers exhibit different affinities for NTE nd AChE (Bertolazzi et al., 1991). Furthermore, metabolic differ- nces between these two species could favor a lower metabolism f the enantiomer with apparently much greater affinity for NTE in umans, and the opposite could be true in hens (Battershill et al., 004). Thus, the aim of this study was to evaluate, in the blood and rain of hens, in the blood of humans, and in SH-SY5Y human neu- oblastoma cells the potential of the methamidophos enantiomers o induce delayed neurotoxicity using the ratio between NTE inhi- ition and AChE inhibition as a possible indicator. Mipafox was also sed as a positive control because it is known as a compound that nduces OPIDN. In addition, reference values for LNTE and AChE n erythrocytes are presented in a sample of donors not exposed o pesticides. Calpain activation was also evaluated because it has een suggested as contributor to OPIDN (El-Fawall et al., 1990; lynn, 2000; Choudhary and Gill, 2001; Emerick et al., 2010). . Materials and methods .1. Chemicals Sodium dodecyl sulfate (SDS), paraoxon, bovine serum albumin (BSA), oomassie Brilliant Blue G-250, Histopaque-1077, tris(hydroxymethyl) minomethane, ethylenediaminetetraacetic acid (EDTA), phosphoric acid 85%, cetylthiocholine (ACTh) and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) were pur- hased from Sigma, St. Louis, MO, USA; mipafox and phenyl valerate were obtained rom Oryza Laboratories, Inc., Chelmsford, MA, USA; sodium citrate and triton X-100 y 292 (2012) 145– 150 were purchased from Rhiedel-de Haën, Hannover, Germany; 4-aminoantipyrine, potassium ferricyanide, and dimethylformamide were purchased from Merck, Darmstadt, Germany; heparin 25,000 IU/5 ml was obtained from Roche, Rio de Janeiro, Brazil; Deltametrin (K-otrine®) was obtained from Bayer Cropscience Ltd., Rio de Janeiro, RJ, Brazil; and piperazine citrate (Proverme®) was purchased from Tortuga Agrarian Zootechnical Company, São Paulo, Brazil. The analytical standard (±)-methamidophos was obtained from Sigma, St. Louis, MO, USA, and the enantiomeric separation was conducted according to the method described by Emerick et al. (2011). The enantiomers of methamidophos were obtained with 99.5% of optical purity for the (+)-methamidophos and 98.3% of optical purity for the (−)-methamidophos. Initially, mipafox was prepared at 0.1 mM concentration level, (+)-methamidophos was prepared at 1000 mM concentration level and (−)-methamidophos was prepared at 10,000 mM concentration level. All these solutions were prepared in absolute ethanol. These concentrates were then diluted at least 100× for incubation with neuroblastoma cells and other tissues to obtain a final concentration of 1% for ethanol. This solvent was chosen based on methamidophos solubility and on previous work that employed SH-SY5Y cells (Ehrich et al., 1997). All other chemicals employed in this study were of analytical grade. 2.2. Animals Twelve isabrown leghorn hens (aged 70–90 weeks, weighing 1.5–2.0 kg) were obtained from the Hayashi farm cooperative of Guatapará, SP, Brazil. Before the experiments were initiated, the hens were treated to eliminate ecto-parasites and endo-parasites, as described elsewhere (DeOliveira et al., 2002; Emerick et al., 2010). After this treatment (1 month), the hens were housed at a density of 3 per cage in a temperature- and humidity-controlled room (24 ± 2 ◦C and 55% ± 10 RH) on an automatic 12:12 light–dark photocycle with lights activated at 8 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 24/2009). 2.3. Human volunteers Blood was collected from 80 volunteers at the hemocenter of the School of Pharmaceutical Sciences of Araraquara – UNESP, SP, Brazil. Donors were invited to participate in this study after undergoing the standard screening required of all blood donors, and, after this first step, the purpose of this study was explained to them. After declaring that they accepted the terms of participation in the study, volunteers were invited to sign the Form of Consent and Statement of Grant for Biological Material that are requirements of 196/1996 Resolution of the Brazilian National Health Council. In addition to the various requirements that a blood donor must satisfy, we applied a questionnaire prior to screening to investigate the vol- unteers’ habits. We asked the following key questions: Do you smoke? Are you taking any medicine? Did you drink any alcoholic beverages in the last two days? Did you have some contact with pesticides in the last 30 days? These questions were applied to reduce confounding factors. Next, an employee of the hemocenter collected approximately 5 ml of blood in heparinized tubes for vacuum collection. All of these 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 humans in research (Reso- lution 09/2009). 2.4. Cell culture SH-SY5Y human neuroblastoma cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Passages 10–22 were used for these experiments. The human cells were grown in 15–20 ml F12 nutrient mixture (F12 HAM; Sigma Cell Culture, St. Louis, MO) containing 15% fetal bovine serum (FBS; Summit Biotechnology, FL Collins, CO) and 1% of an antibiotic–antimycotic solu- tion (10,000 IU/ml penicillin, 10,000 �g/ml streptomycin, 25 �g/ml amphotericin B, Mediatech Inc., Manassas, VA) in 225-cm2 flasks (Coming Costar Corporation, Cambridge, MA). Previous studies determined that these media provided optimal esterase activities for these cell lines (Ehrich et al., 1995). Cells were observed daily. To induce differentiation and maximize basal AChE activity, SH-SY5Y human neuroblastoma cells were treated with 10 �M retinoic acid when reaching 60–80% confluency. The SH-SY5Y cells remained in the retinoic acid-containing medium for 4 days before being harvested. To harvest SH-SY5Y cells, the medium was removed and the cells incubated in 3.0 ml of trypsin 0.5% (diluted in medium) for 5 min before being removed from the flask by pipetting. After harvesting, viability was deter- mined by trypan blue exclusion to be >80%. Following centrifugation, the cells were resuspended in PBS at a concentration of 1 × 107 cells/ml and kept with the inhibitors for one hour before assays. 2.5. Sample collection For determination of LNTE activity, 2.5 ml of blood were collected from the axil- lary veins of the hens in 3-ml syringes already containing 0.1 ml of heparin per ml icolog o o t e s ( 1 0 p w u p f b o n 2 H a a T u ( S E i f 6 s a e a d w m w o N i 2 d c r t f T O t d d 3 3 a b c b L d T p m t a G.L. Emerick et al. / Tox f blood (5000 IU/ml diluted 1/5 with 0.9% saline solution). For the determination f AChE and NTE activity in the brain of the hens, they were sacrificed by cervical orsion followed by decapitation. Next, a small amount (about 0.4 g) of tissue was xtracted from the frontal part of the brain. This tissue was homogenized in the odium phosphate buffer (0.1 M, pH 8.0) for the AChE assay and in the Tris buffer 50 mM Tris–HCl, 0.2 mM EDTA, pH 8.0, 25 ◦C) for the NTE assay at a concentration of g tissue to 20 ml of buffer. To measure the activity of AChE in human erythrocytes, .5 ml of whole blood was extracted, and erythrocytes were separated from the lasma by centrifugation (500 × g, 10 min). These erythrocytes were subsequently ashed twice with 1.5 ml (3 times the volume of blood) of isotonic saline solution sing the same spin cycle for plasma separation to avoid interference from other lasma esterases. After this step, the erythrocytes were diluted 1/600 in water for urther analysis. For the determination of the LNTE activity of humans, 2.5 ml of lood was collected, as described above for the hens. Fifty microliters of 1 × 107/ml f cells were used as sample for the determinations of AChE and NTE in the human euroblastoma cells. .6. Enzyme assays To assay the LNTE activity, the lymphocytes were separated from the blood using istopaque-1077® according to the Sigma diagnostic procedure. The lymphocytes nd brains were diluted in a buffer (50 mM Tris–HCl, 0.2 mM EDTA, pH 8.0, 25 ◦C) nd their protein concentrations were determined by the method of Bradford (1976). he NTE and LNTE activity were assayed, as described by Correll and Ehrich (1991) sing phenyl valerate as substrate. In addition, in the same volume of the sample 50 �L), 6 different concentrations of the OPs (ranging from 0.01 to 100 mM, see ection 2.1) were employed. The incubations were done for 1 h, at 37 ◦C. The activity of cholinesterases was determined using the method described by llman et al. (1961), with 6 different concentrations of the OPs as inhibitors (rang- ng from 0.0001 to 10 mM in ethanol, see Section 2.1). The incubations proceeded or 1 h, at 37 ◦C. Four readings of each concentration were recorded at intervals of 0 s at 37 ◦C and 450 nm with constant stirring at 600 rpm in a UV/visible HP 8453 pectrophotometer. The absorbance used to calculate the enzyme activity was the verage per min of these 4 readings. The concentrations of protein samples were valuated using the Bradford method (1976) before the enzyme evaluations because ll of the enzyme activities were reported in terms of �mol/min/g of protein. Calpain activity in the chicken brain and neuroblastoma cells was analyzed as escribed elsewhere (Emerick et al., 2010), but before the assay, tissue homogenate as incubated with mipafox (0.01 mM) or (+)-methamidophos (10 mM) or (−)- ethamidophos (100 mM) for one hour, at 37 ◦C. CaCl2 in a concentration of 4 mM as added in the follow proportion: 1 g of tissue or 1 ml of cells (1 × 107/ml)/0.01 ml f OP in ethanol/1 ml of CaCl2. The concentrations of OPs used were based on the TE inhibition with concentration for each compound causing at least 80% NTE nhibition. .7. Statistical analyses Inhibitor concentrations capable of inhibiting 50% of enzyme activity (IC50) were etermined using the equation of the line graph of the log of % activity versus the oncentration of inhibitor (semilog plots). The semilog plots are not shown to avoid epetitions of results. The regression coefficients of these lines were calculated using he method of least squares. Differences in biochemical analyses were examined or statistical significance by one way ANOVA (Analysis Of Variance) followed by ukey’s test for multiple comparisons. These tests were performed in Microsoft ffice Excel 2007 for Windows. The definition of significance was p < 0.05 for all sta- istical analyses. All biochemical data are presented as the averages of three samples one in triplicate (n = 3). All biochemical data are expressed as means ± the standard eviation (SD). . Results .1. Neuropathy target esterase Control values for NTE and AChE activities in hens and humans re presented in Table 1. All of the coefficients of variation remained elow 20%. AChE activity was not evaluated in the hens’ erythro- ytes because a previous study showed that this activity could not e detected (Wilson and Henderson, 1992). The potencies of the isomers of methamidophos against NTE and NTE differed. The inhibition curves of NTE in hens and humans are epicted in Fig. 2A, C, E and G and IC50 values are reported in Table 2. hese data indicate that the (+)-methamidophos form was a more otent inhibitor of NTE than the (−)-methamidophos form. The (−)- ethamidophos isomer exhibited an IC50 value approximately 5.6 imes greater than did the (+)-methamidophos isomer for the LNTE ctivity of hen and approximately 4 times that observed for the y 292 (2012) 145– 150 147 inhibition of human LNTE activity. The percentage activity versus inhibitor concentrations exhibited high inverse regression coeffi- cients for all NTE activities (Table 2). For the NTE in the hen brain and in the neuroblastoma cells the (−)-methamidophos isomer exhibited IC50 values approximately 41 and 160 times greater than did the (+)-methamidophos isomer (Table 2). Mipafox had a lower IC50 value for the hen brain and for the SH-SY5Y cells when com- pared to the isoforms of methamidophos (Fig. 2H and Table 2). Comparing the results of IC50 values for both species, it was possi- ble to see that human cells (SH-SY5Y and lymphocytes) are more sensitive to the methamidophos enantiomers compared to tissues from hens. This was not true, however, for mipafox, as hen brain was more sensitive than SH-SY5Y cells (Fig. 2H). 3.2. Acetylcholinesterase The curves of inhibition for AChE in the brain of hens are depicted in Fig. 2D and indicate that the isoform (−)- methamidophos was a more potent enzyme inhibitor than the (+)-methamidophos form. Human AChE in SH-SY5Y cells and ery- throcytes (Fig. 2B and F) presented similar behavior to that of AChE in hen brains with the (−)-methamidophos form a more potent inhibitor than the (+)-methamidophos. The (+)-methamidophos isomer exhibited an IC50 value approximately 7 times greater than that of the (−)-methamidophos isomer for the inhibition of AChE in hen brain (Table 2). The lines of the log of percentage activity versus inhibitor concentration demonstrated strong inverse regres- sion coefficients in all tissue tested (Table 2). Mipafox was used as a known inducer of OPIDN and presented a lower IC50 value for the chicken brain and an intermediate IC50 value for the SH-SY5Y cells compared to the isoforms of methamidophos (Fig. 2H and Table 2). Comparing the results of IC50 values for both species, it was noted that human cells (SH-SY5Y and erythrocytes) are more sensitive to the compounds tested in relation to hen tissues. These results are summarized in Table 2. The ratios of enzyme IC50 values pre- sented in Table 2 show that the isoforms of methamidophos are stronger inhibitors for AChE than NTE. On the other hand, mipafox is a stronger inhibitor of NTE. 3.3. Calpain Calpain activation was evaluated in hen brain and in the SH-SY5Y neuroblastoma cells. Although (+)-methamidophos expo- sure resulted in a small calpain activation, neither enantiomer of methamidophos was able to produce activation of calpain statisti- cally different from control. In contrast, mipafox was able to induce a 5% increase in the calpain activity in hen brain and a 15% increase in the human cells (Fig. 3). 4. Discussion The results of the present study demonstrated differences between the enantiomers of methamidophos in their ability to inhibit both NTE and AChE. This study also demonstrated that these differences could be determined in vitro. Enantioseparation has become an important tool in the discernment of the actual toxic agent responsible for a particular purpose. However, when neurotoxicity studies in animals require large quantities of the compounds in question, an initial in vitro screening is useful. Although the sensitivities of NTE and AChE may be similar in hens and humans when a racemic mixture is tested, our results with the enantiomers of the methamidophos demonstrated that they may not be predicted or extrapolated from one species to another. One explanation may relate to metabolic differences between species. Methamidophos can cause a cholinergic crisis in hens so strong that it will be lethal before the onset of clinical signs of 148 G.L. Emerick et al. / Toxicology 292 (2012) 145– 150 Table 1 Summary of the normal activity of the enzymes NTE and AChE in human and hen tissues. Assessed enzyme Mean (�mol/min/g of protein) Standard deviation Coefficient of variation LNTE in humans 7.0 1.2 17.1 NTE in SH-SY5Y cells 19.9 3.7 18.6 LNTE in hens 9.4 1.8 19.1 NTE in hen brains 29.3 4.4 15.1 AChE in human erythrocytes 6.9 1.3 18.8 AChE in SH-SY5Y cells 82.9 12.8 15.4 AChE in hen brains 978.2 101.4 10.4 n = 12 for hens and SH-SY5Y cells and 80 for humans. Each sample was assayed in triplicate. AChE, acetylcholinesterase; LNTE, lymphocyte neuropathy target esterase. Fig. 2. Comparison among the curves of % of activity versus the concentrations of mipafox, (+)-methamidophos and (−)-methamidophos for NTE and AChE in hens and humans. Results for methamidophos are in (A)–(G); results for mipafox are in (H). Each point was obtained by averaging three samples. Each sample was analyzed in triplicate (n = 3). IC50 values and their SD are given in Table 2. G.L. Emerick et al. / Toxicology 292 (2012) 145– 150 149 Table 2 Summary of the IC50 values and R-squared of NTE and AChE in human and hen tissues. Assessed enzyme Inhibitor IC50 (mM) R-squared Ratio IC50 NTE/IC50 AChE LNTE of humans (+)-Methamidophos 0.862 ± 0.0710* 0.9725 0.55 (−)-Methamidophos 3.43 ± 0.252 0.9278 14.8 LNTE of hens (+)-Methamidophos 1.32 ± 0.202* 0.9385 (−)-Methamidophos 7.42 ± 0.285 0.9744 NTE in the brain of hens Mipafox 0.0027 ± 0.0008 0.9899 0.13 (+)-Methamidophos 0.550 ± 0.0926* 0.9731 0.23 (−)-Methamidophos 22.8 ± 3.24 0.9767 66.1 NTE in SH-SY5Y cells Mipafox 0.0193 ± 0.0015 0.9538 1.27 (+)-Methamidophos 0.0892 ± 0.0221* 0.9536 3.96 (−)-Methamidophos 14.3 ± 2.32 0.9401 3325 AChE in the brain of hens Mipafox 0.0206 ± 0.0025 0.9720 (+)-Methamidophos 2.42 ± 0.0091* 0.9814 (−)-Methamidophos 0.345 ± 0.0112 0.9661 AChE in SH-SY5Y cells Mipafox 0.0152 ± 0.0017 0.9926 (+)-Methamidophos 0.0225 ± 0.0031* 0.9543 (−)-Methamidophos 0.0043 ± 0.0010 0.9364 AChE in human erythrocytes (+)-Methamidophos 1.58 ± 0.061* 0.9683 (−)-Methamidophos 0.231 ± 0.0121 0.9246 IC50 values are represented as means ± SD. These results were obtained with the means of three concentration-response curves. AChE, acetylcholinesterase; LNTE, lymphocyte neuropathy target esterase; IC50, inhibitory concentration for 50% of enzymatic activity. midop c O f e o l i s h i t 1 t a p F ( n a s * A statistically significant difference compared with the other isomer of metha omparisons). PIDN. Therefore, in hens, the enantiomer with a higher affinity or AChE may be less metabolized than in other species, and the nantiomer that exhibits greater affinity for NTE may be less metab- lized in humans. Studies done only with tissue from hens could ead to the erroneous conclusion that methamidophos does not nduce OPIDN in humans. Therefore, the combination of in vitro tudies on human and hen enzymes and studies of metabolism in ens could predict whether the OP is capable of generating OPIDN n both species (Battershill et al., 2004). There are several research studies that describe calpain activa- ion in hens after intoxication by a neuropathic OP (El-Fawall et al., 990; Choudhary and Gill, 2001; Emerick et al., 2010). In Wallerian- ype degeneration an excessive intake of calcium by the cell can ctivate calpain. This enzyme promotes digestion of the terminal ortion of axons, preventing the transmission of nerve impulses to ig. 3. Calpain activation caused by mipafox (0.01 mM), (+)-methamidophos 10 mM) and (−)-methamidophos (100 mM) in the hen brain and SH-SY5Y human euroblastoma cells after 1 h of incubation at 37 ◦C. Each point was obtained by veraging three samples. Each sample was analyzed in triplicate (n = 3). *Values tatistically different from controls (p < 0.05). hos (p < 0.05, according to one way ANOVA followed by Tukey’s test for multiple the post-synaptic cells (Moser et al., 2007). In the present work, an in vitro calpain assay demonstrates that only mipafox was able to promote calpain activation. This effect was greater with human neuroblastoma cells, probably because they are relatively pure compared to the multiple cell types found in a brain homogenate. An early study by Ehrich et al. (1997) showed that capability to cause or not cause OPIDN could be predicted by ratios of the IC50 values in human and mouse neuroblastoma cells. Later, Sogorb et al. (2010) proposed an alternative methodology to predict whether an OP is able to induce OPIDN. This method is based on the compari- son of the in vitro inhibition (and aging of NTE) of both enzymes (NTE and AChE) in human and hen cells. The authors tested 10 OPs (6 neuropathic and 4 non-neuropathic), and stated that if the IC50NTE/IC50AChE ratio is greater than five, then the compounds would not be able to induce the neuropathy. This was because the concentrations necessary for inhibition and aging of greater than 70% of NTE would not be compatible with the survival of individ- uals due to strong cholinergic crisis before the onset of delayed effects. However, if the IC50NTE/IC50AChE ratio is less than five, the OP may be a neuropathic compound if it has the ability to induce the “aging” reaction. Applying this hypothesis to the results of this in vitro study, we conclude that the (−)-methamidophos form would not be able to generate OPIDN in humans and hens, even if the aging reaction of NTE was to occur. However, other vari- ables exist in vivo, such as differences in metabolism. Aging studies were not performed because prior works demonstrate that both enantiomers of methamidophos are weak inducers of NTE aging and that greater than 90% inhibition of NTE is required to induce OPIDN in vivo (Vilanova et al., 1987; Johnson et al., 1989; Sogorb et al., 1997; Kellner et al., 2000). However, the aging protocol is essential to make conclusions based on in vitro tests in an unknown chiral organophosphate. Previous experiments using different species have demon- strated toxicological differences between the stereoisomers of methamidophos, noting differences in the potential to induce OPIDN (Senanayake and Johnson, 1982; Lotti et al., 1995; McConnell et al., 1999; Battershill et al., 2004). Using brain from human and hen Bertolazzi et al. (1991) examined the ratio between the inhibition constant of AChE and the inhibition constant of NTE. 1 icolog T t t h i d t i A t i e a f m t i h b h e b F b t h w N C A d 2 t o A R B B B B C C D E 50 G.L. Emerick et al. / Tox he authors observed, as did the present study with IC50 values, that he ki AChE/ki NTE ratio of (−)-methamidophos was much higher han that observed for the other isomer. Thus, the most probable ypothesis is that the (+)-methamidophos form can induce OPIDN n humans and hens. However, further studies are necessary to etermine if differences between the two species in their ability o induce OPIDN is related to metabolism or to the enantioselectiv- ty of these compound for inhibiting and aging NTE and inhibiting ChE activities. In conclusion, significant differences were observed between he IC50 values of the three isoforms of methamidophos regard- ng their in vitro inhibition of the activities of the NTE and AChE nzymes. The (−)-methamidophos form exhibited an IC50 value pproximately 6 times greater than did the (+)-methamidophos orm in inhibiting LNTE activity in chickens, and the (+)- ethamidophos form demonstrated a IC50 value approximately 7 imes greater than that of the (−)-methamidophos form in inhibit- ng hen AChE activity. Differences between species were noted, as uman esterases showed more sensitivity than hen esterases to oth enantiomers. The model of SH-SY5Y human cells showed the igher difference between the NTE inhibition of methamidophos nantiomers and the hen brain showed the higher difference etween the AChE inhibition of methamidophos enantiomers. inally, considering only the in vitro results (NTE and AChE inhi- ition), the (+)-methamidophos form exhibited a greater potential o induce OPIDN than did the (−)-methamidophos form both for umans and for hens. However, this potential in inducing OPIDN as lower than the potential observed with mipafox considering TE and AChE inhibition and calpain activation as indicators. onflict of interest There are no conflicts of interest. cknowledgements Financial support for this study was provided by the “Fundaç ão e Amparo à Pesquisa do Estado de São Paulo” – FAPESP Grant # 009/51048-8 and by the Fundunesp Proc. 01318/10 DFP. Addi- ional funding was provided by Virginia-Maryland Regional College f Veterinary Medicine. Technical assistance was provided by Maria parecida dos Santos, Kristel Fuhrman and Melissa Makris. eferences attershill, J.M., Edwards, P.M., Johnson, M.K., 2004. Toxicological assessment of isomeric pesticides: a strategy for testing of chiral organophosphorus (OP) com- pounds for delayed polyneuropathy in a regulatory setting. Food Chem. Toxicol. 42, 1279–1285. ertolazzi, M., Caroldi, S., Moretto, A., Lotti, M., 1991. Interaction of methamidophos with hen and human acetylcholinesterase and neuropathy target esterase. Arch. Toxicol. 65, 580–585. radford, M.M., 1976. A rapid and sensitive method for the quantification of micro- gram quantity of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. RASIL. Conselho Nacional de Saúde. Resoluç ão n◦ 196, de 10 de outubro de 1996. Dispõe sobre aprovar as diretrizes e normas regulamentadoras de pesquisas envolvendo seres humanos. Brasília, DF. houdhary, S., Gill, K.D., 2001. Protective effect of nimodipine on dichlorvos-induced delayed neurotoxicity in rat brain. Biochem. Pharmacol. 62, 1265–1272. orrell, L., Ehrich, M., 1991. A microassay method for neurotoxic esterase determi- nations. Fundam. Appl. Toxicol. 16, 110–116. eOliveira, G.H., Moreira, V., Goes, S.P.R., 2002. Organophosphate induced delayed neuropathy in genetically dissimilar chickens: studies with tri-ortho-cresyl phosphate (TOCP) and trichlorfon. Toxicol. Lett. 136, 143–150. hrich, M., Correll, L., Carlson, K., Wilcke, J., Veronesi, B., 1995. Examination of culture conditions on esterase activities in human and mouse neuroblastoma cells. In Vitro Toxicol. 8, 199–207. y 292 (2012) 145– 150 Ehrich, M., Correll, L., Veronesi, B., 1997. Acetylcholinesterase and neuropathy target esterase inhibitions in neuroblastoma cells to distinguish organophosphorus compounds causing acute and delayed neurotoxicity. Fundam. Appl. Toxicol. 38, 55–63. El-Fawal, H.A.N., Correll, L., Gay, L., Ehrich, M., 1990. Protease activity in brain, nerve and muscle of hens given neuropathy-inducing organophosphates and a calcium channel blocker. Toxicol. Appl. Pharmacol. 130, 133–142. Ellman, G.L., Courtney, K.D., Andres, V., Featherstone, R.M., 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharma- col. 7, 88–95. Emerick, G.L., Peccinini, R.G., DeOliveira, G.H., 2010. Organophosphorus-induced delayed neuropathy: a simple and efficient therapeutic strategy. Toxicol. Lett. 192, 238–244. Emerick, G.L., Oliveira, R.V., Belaz, K.R.A., Gonç alves, M., Deoliveira, G.H., 2011. Semipreparative enantioseparation of methamidophos by hplc–uv and prelim- inary in vitro study of butyrylcholinesterase inhibition. Environ. Toxicol. Chem., doi:10.1002/etc.729. Fedalei, A., Nardone, R.M., 1983. An in vitro alternative for testing the effect of organophosphates on neurotoxic esterase activity. In: Goldberg, A. (Ed.), Alter- native Methods in Toxicology, Product Safety Evaluation, vol. 1. Mary Ann Liebert Inc., New York, pp. 253–269. Glynn, P., 2000. Neural development and neurodegeneration: two faces of neuropa- thy target esterase. Prog. Neurobiol. 61, 61–74. Johnson, M.K., 1975. Structure–activity relationships for substrates and inhibitors of hen brain neurotoxic esterase. Biochem. Pharmacol. 24, 797–805. Johnson, M.K., 1981. Delayed neurotoxicity-Do trichlorphon and/or dichlorvos cause delayed neuropathy in man or in test animals? Acta Pharmacol. Toxicol. 49S, 87–98. Johnson, M.K., 1988. Sensitivity and selectivity of compounds interacting with neu- ropathy target esterase. Biochem. Pharmacol. 37, 4095–4104. Johnson, M.K., Vilanova, E., Read, D.J., 1989. Biochemical and clinical tests of the delayed neuropathic potential of some O-alkylO-dichlorophenyl phospho- ramidate analogues of methamidophos (O,S-dimethyl phosphorothioamidate). Toxicology 54, 89–100. Johnson, M.K., Vilanova, E., Read, D.J., 1991. Anomalous biochemical responses in tests of the delayed neuropathic potential of methamidophos (O,S-dimethyl phosphorothioamidate), its resolved isomers and of some higher O-alkyl homo- logues. Arch. Toxicol. 65, 618–624. Johnson, M.K., Jacobsen, D., Meredith, T.J., Eyer, P., Heath, A.J., Ligtenstein, D.A., Marrs, T.C., Szinicz, L., Vale, J.A., Haines, J.A., 2000. Evaluation of antidotes for poisoning by organophosphorus pesticides. Emerg. Med. 12, 22–37. Jortner, B.S., Hancock, S.K., Hinckley, J., Flory, L., Colby, L., Tobias, L., Williams, L., Ehrich, M., 2005. Neuropathological studies of rats following multiple expo- sure to tri-ortho-tolyl phosphate, chlorpyrifos and stress. Toxicol. Pathol. 33, 378–385. Kellner, T., Sanborn, J., Wilson, B., 2000. In vitro and in vivo assessment of the effect of impurities and chirality on methamidophos-induced neuropathy target esterase aging. Toxicol. Sci. 54, 408–415. Lin, K., Zhou, S., Xu, C., Liu, W., 2006. Enantiomeric resolution and biotoxicity of methamidophos. J. Agric. Food Chem. 54, 8134–8138. Lotti, M., 1992. The pathogenesis of organophosphate polyneuropathy. Crit. Rev. Toxicol. 21, 465–487. Lotti, M., Moretto, A., Bertolazzi, M., Peraica, M., Fioroni, F., 1995. Organophosphate polyneuropathy and neuropathy target esterase: studies with methamidophos and its resolved optical isomers. Arch. Toxicol. 69, 330–336. McConnell, R., Téllez, E.D., Cuadra, R., Torres, E., Keifer, M., Almendárez, J., Miranda, J., El-Fawal, H.A.N., Wolff, M., Simpsom, D., Lundberg, I., 1999. Organophos- phate neuropathy due to methamidophos: biochemical and neurophysiological markers. Arch. Toxicol. 73, 296–300. Moser, V.C., Aschner, M., Richardson, R.J., Philbert, M.A., 2007. Toxic responses of the nervous system. In: Klaassen, C.D. (Ed.), Casarett and Doull’s Toxicology: The Basic Science of Poisons. McGraw-Hill, New York, pp. 631–664. OECD Guidelines for the Testing of Chemicals, 1995. Test No. 418: Delayed Neuro- toxicity of Organophosphorus Substances Following Acute Exposure. OECD Guidelines for the Testing of Chemicals, 1995. Test No. 419: Delayed Neuro- toxicity of Organophosphorus Substances: 28-day Repeated Dose Study. Senanayake, N., Johnson, M.K., 1982. Acute polyneuropathy after poisoning by a new organophosphate insecticide. N. Engl. J. Med. 306, 155–157. Sogorb, M.A., Díaz-Alejo, N., Pellín, M.C., Vilanova, E., 1997. Inhibition and aging of neuropathy target esterase by the stereoisomers of a phosphoramidate related to methamidophos. Toxicol. Lett. 93, 95–102. Sogorb, M.A., González-González, I., Pamies, D., Vilanova, E., 2010. An alternative in vitro method for detecting neuropathic compounds based on acetyl- cholinesterase inhibition and on inhibition and aging of neuropathy target esterase (NTE). Toxicol. In Vitro 24, 942–952. Vilanova, E., Johnson, M.K., Vicedo, J.L., 1987. Interaction of some unsubstituted phosphoramidate analogs of methamidophos (O,S-dimethyl phosphoroth- ioamidate) with acetylcholinesterase and neuropathy target esterase of hen brain. Pestic. Biochem. Physiol. 28, 224–238. Wilson, B.W., Henderson, J.D., 1992. Blood esterase determinations as markers of exposure. Rev. Environ. Contam. Toxicol. 128, 55–69. dx.doi.org/10.1002/etc.729 Comparative in vitro study of the inhibition of human and hen esterases by methamidophos enantiomers 1 Introduction 2 Materials and methods 2.1 Chemicals 2.2 Animals 2.3 Human volunteers 2.4 Cell culture 2.5 Sample collection 2.6 Enzyme assays 2.7 Statistical analyses 3 Results 3.1 Neuropathy target esterase 3.2 Acetylcholinesterase 3.3 Calpain 4 Discussion Conflict of interest Acknowledgements References