Acetylcholinesterase and Neuropathy Target Esterase Inhibitions in Neuroblastoma Cells to Distinguish Organophosphorus Compounds Causing Acute and Delayed Neurotoxicity

FUNDAMENTAL AND APPLIED TOXICOLOGY ARTICLE NO.

38, 55–63 (1997)

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Acetylcholinesterase and Neuropathy Target Esterase Inhibitions in Neuroblastoma Cells to Distinguish Organophosphorus Compounds Causing Acute and Delayed Neurotoxicity1 Marion Ehrich,*,2 Linda Correll,* and Bellina Veronesi† *Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg, Virginia 24061; and †Neurotoxicology Division, United States Environmental Protection Agency, Research Triangle Park, North Carolina 27711 Received November 18, 1996; accepted May 29, 1997

Organophosphorus (OP) insecticides, lubricants, and plasticizers are a potential health and safety hazard to individuals associated with their manufacture, formulation, application, and field use (Marrs, 1993; Chambers and Levi, 1992). OPs used as insecticides inhibit acetylcholinesterase (AChE), the enzyme responsible for degradation of the neurotransmitter acetylcholine. In addition, some OPs can inhibit neuropathy target esterase (NTE, neurotoxic esterase), an enzyme which is associated with organophosphorus-induced delayed neuropathy (OPIDN), a progressive disorder which does not appear until weeks after inhibition of NTE (Ecobichon, 1991; Johnson, 1982). Although the precise mechanism(s) of OPIDN is yet undetermined, sufficient NTE must be essentially irreversibly inhibited before it develops. Man and certain animal species (e.g., hen, cat, and sheep) are highly susceptible to clinical manifestations of OPIDN (Johnson, 1982). NTE is, however, also inhibited in species which are less likely to display clinical and neuropathological changes after OP administration (e.g., rats and mice) (Ehrich et al., 1995a; Veronesi et al., 1991). Current federal testing requirements for OP insecticides include dose-related inhibitions of AChE and NTE in exposed animals (US EPA, 1991). Since these esterases are also present in neuroblastoma cells (Fedalei and Nardone, 1983; Carrington et al., 1985; Nostrandt and Ehrich, 1992, 1993; Veronesi and Ehrich, 1993a,b; Ehrich et al., 1994, 1995b), we have explored the possibility of using cell lines to assess OP neurotoxicity potential. Our previous studies indicated that neuropathic OPs (i.e., those causing OPIDN) could be identified by NTE inhibition in human neuroblastoma cells (Ehrich et al., 1994). The present study expands this work to include a comparison of concentrationdependent inhibitions of both NTE and AChE in cell lines originating from both human and rodent sources. OPs that are more likely to cause cholinergic crisis (a result of AChE inhibition) and neuropathic OPs (those associated with OPIDN) were used as test compounds.

Acetylcholinesterase and Neuropathy Target Esterase Inhibitions in Neuroblastoma Cells to Distinguish Organophosphorus Compounds Causing Acute and Delayed Neurotoxicity. Ehrich, M., Correll, L., and Veronesi, B. (1997). Fundam. Appl. Toxicol. 38, 55–63. The differential inhibition of the target esterases acetylcholinesterase (AChE) and neuropathy target esterase (NTE, neurotoxic esterase) by organophosphorus compounds (OPs) is followed by distinct neurological consequences in exposed subjects. The present study demonstrates that neuroblastoma cell lines (human SHSY5Y and murine NB41A3) can be used to differentiate between neuropathic OPs (i.e., those inhibiting NTE and causing organophosphorus-induced delayed neuropathy) and acutely neurotoxic OPs (i.e., those highly capable of inhibiting AChE). In these experiments, concentration–response data indicated that the capability to inhibit AChE was over 1001 greater than the capability to inhibit NTE for acutely toxic, nonneuropathic OPs (e.g., paraoxon and malaoxon) in both cell lines. Inhibition of AChE was greater than inhibition of NTE, without overlap of the concentration– response curves, for OPs which are more likely to cause acute, rather than delayed, neurotoxic effects in vivo (e.g., chlorpyrifosoxon, dichlorvos, and trichlorfon). In contrast, concentrations inhibiting AChE and NTE overlapped for neuropathy-causing OPs. For example, apparent IC50 values for NTE inhibition were less than 9.6-fold the apparent IC50 values for AChE inhibition when cells were exposed to the neuropathy-inducing OPs diisopropyl phosphorofluoridate, cyclic tolyl saligenin phosphate, phenyl saligenin phosphate, mipafox, dibutyl dichlorovinyl phosphate, and di-octyl-dichlorovinyl phosphate. In all cases, esterase inhibition occurred at lower concentrations than those needed for cytoxicity. These results suggest that either mouse or human neuroblastoma cell lines can be considered useful in vitro models to distinguish esterase-inhibiting OP neurotoxicants. q 1997 Society of Toxicology.

1 These data were presented at the 1996 Annual Meeting of the Society of Toxicology, March 1996, Anaheim, CA, and published in abstract form (Fundam. Appl. Toxicol. 30S, 310, 1996). 2 To whom correspondence should be addressed. Fax: (540) 231-7367.

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0272-0590/97 $25.00 Copyright q 1997 by the Society of Toxicology. All rights of reproduction in any form reserved.

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MATERIALS AND METHODS Chemicals. Esterase inhibitors included paraoxon (O,O-diethyl 4-nitrophenyl phosphate; from Chem Services, West Chester, PA; 98% pure), malaoxon (S-(1,2-dicarbethoxyethyl) O,O-dimethyl phosphorothiolate; from American Cyanamid Co., Pearl River, NY; 92% pure), cyclic tolyl saligenin phosphate (TSP; from Lark Enterprises, Webster, MA; 100% pure), chlopyrifos-oxon (O,O-diethyl O-(3,5,6-trichloro-2-pyridyl) phosphate; from DowElanco, Indianapolis, IN; 99% pure), diisopropyl phosphorofluoridate (DFP; from Aldrich Chemical Co., Milwaukee, WI; 100% pure), mipafox (N*,N*bis(1-methylethyl)-phosphordiamidic fluoride; from Lark Enterprises, 100% pure), cyclic phenyl saligenin phosphate (PSP; from Lark Enterprises, 100% pure), dichlorvos (O,O-dimethyl O-(2,2-dichlorovinyl) phosphate, also known as DDVP; from Fermenta Animal Products, Kansas City, MO; 96% pure), O,O-dibutyl O-(2,2-dichlorovinyl) phosphate (DBVP; from Oryza Laboratories, Chelmsford, MA; 87% pure), O,O-di-octyl O-(2,2-dichlorovinyl) phosphate (DOVP; from Oryza Laboratories, 45% pure, 39% non-esterase inhibiting dioctylphosphite), and trichlorfon (O,O-dimethyl 1-hydroxy-2,2,2-trichloroethylphosphonate; from Miles Agricultural Services, 99% pure). Test agent purity was provided by the supplier (paraoxon, malaoxon, chlorpyrifos-oxon, TSP, DFP, and trichlorfon) or determined by GC-MS analyses done at Virginia Tech (dichlorvos, PSP, mipafox, DBVP, DOVP). Optical isomers were not determined. These test compounds included OPs more likely to cause cholinergic crisis than OPIDN (e.g., paraoxon, malaoxon, chlorpyrifos-oxon, dichlorvos, and trichlorfon) and OPs associated with the delayed neurotoxicity, OPIDN (e.g., TSP, DFP, PSP, mipafox, DBVP, and DOVP) (Ecobichon, 1991; Richardson, 1995; Ehrich et al., 1995a; Hollinghaus, 1984; Johnson, 1981, 1988; Moretto et al., 1989). Also included was a series of dichlorovinyl congeners reported to have different capabilities to induce OPIDN (dichlorvos, DBVP, and DOVP) (Johnson, 1981; Hollinghaus, 1984). Test compounds were initially prepared as 1001 M concentrates in absolute ethanol. These concentrates were then diluted at least 1001 for incubation with neuroblastoma cells. This solvent was chosen because vehicle controls (1% ethanol in 0.01 M phosphate-buffered saline (PBS), pH 7.4) had esterase activities, trypan blue exclusion, and neutral red retention like those of cells not incubated with any solvent (data not shown). Cell culture. SH-SY5Y human neuroblastoma cells were obtained from the laboratory of Dr. J. R. Perez-Polo (University of Texas, Galveston). NB41A3 mouse neuroblastoma cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD). Passages 10–52 for human cells and 48–63 for mouse cells were used for these experiments. The human cells were grown in 40–50 ml F12 nutrient mixture (F12 HAM; Sigma Cell Culture, St. Louis, MO) containing 15% fetal bovine serum (FBS; Summit Biotechnology, Ft. Collins, CO) in 150- or 225-cm2 flasks (Corning Costar Corporation, Cambridge, MA). Mouse cells were grown in minimum essential medium (MEM) with Earle’s salts and leucine (Sigma Chemical Co.), 10% FBS, and 2 mM glutamine. Previous studies determined that these media provided optimal esterase activities for these cell lines (Ehrich et al., 1995b). Cells were observed daily. To induce differentiation and maximize basal AChE activity, SH-SY5Y human neuroblastoma cells were treated with 20 mM retinoic acid when reaching 60–80% confluency. Retinoic acid treatment did not affect basal AChE levels in mouse NB41A3 cells. The SH-SY5Y cells remained in the retinoic acid-containing medium for 4–5 days before being harvested. To harvest SHSY5Y cells, the medium was removed and the cells incubated in 15–20 ml PBS for 15 min before being removed from the flask by pipetting. NB41A3 mouse neuroblastoma cells were harvested by incubating the cells for 15 min in Puck’s solution with 0.3 g EDTA/liter. Previous experiments indicated that method used for harvesting cells had no effect on esterase activities (Ehrich et al., 1995b). After harvesting, viability was determined by trypan blue exclusion to be ú90%. Following centrifugation, the cells were resuspended in PBS at a concentration of 1 1 107 cells/ml. Esterase determinations. Cells (1 1 107/ml) in 1.5-ml volumes were incubated (5% CO2 in ambient air) for 1 hr at 377C with 0.015 ml of test

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compounds so that final concentrations ranged from 5 1 10011 to 1003 M. Preliminary studies indicated that OP-induced inhibition reached a plateau within 30 min and remained stable for incubation times between 30 and 240 min. At the end of the period of incubation with OPs or buffer, 50 ml of the suspended cells was added to wells of microtiter plates for esterase assays (Correll and Ehrich, 1991). For the NTE assay, a preincubation period of 20 min at 377C with 0.064 mM mipafox and/or 0.5 mM paraoxon preceded a 30min incubation with the phenyl valerate substrate (Lark Enterprises, Webster, MA; 50 ml at 1 mg/ml) in a total volume of 200 mL. Phenyl valerate hydrolysis was linear up to at least 120 min in this incubation system. NTE activity was determined as the difference between the hydrolysis of this substrate in incubates containing mipafox / paraoxon (which inhibits all inhibitable esterases) and incubates containing paraoxon (to inhibit esterases other than NTE), with absorbances at 510 nm compared to a phenol standard curve (Johnson, 1982; Correll and Ehrich, 1991). Microtiter wells for the AChE assay contained cells, 50 ml (0.12 mg) 5,5*-dithiobis(nitrobenzoic acid), and 50 ml (0.065 mg) acetylthiocholine substate. Enzymatic hydrolysis of the substrate was determined by the change in absorbance at 410 nm over a 60-min period in an assay system that was linear at least up to 120 min. Substrate and tissue blanks were included. Each OP was incubated with cells on at least three separate occasions, with triplicate wells for each esterase determination run each day. Esterase activities were compared to wells that contained all ingredients except the test OP. Results were expressed as the percentage of the esterase activity of these control cells and analyzed statistically (see below). Cytotoxicity determinations. Cytotoxic effects OPs were determined by a modified neutral red assay (Veronesi and Ehrich, 1993a). This microtiter plate assay of multiple samples uses a dye that is retained by live cells. For this procedure, neuroblastoma cells were harvested, checked for viability using trypan blue exclusion, resuspended in the growth medium described above, and seeded into 96-well plates (200 ml/well of cells at 1 1 105 cells/ml density). Cells were incubated for 2–3 days, until 60–80% confluency was reached. At that time, fresh medium (180 mL) was added along with OP solution (10 ml of a 101 concentration in 10% ethanol to provide no more than 1% vehicle in the final incubate). Final concentrations of OPs were 1003, 1004, and 1005 M. Cells were incubated with OPs for exposure times that ranged from 1 to 72 hr. At the end of the incubation period, the OP-containing medium was pipetted from the wells, and the cells adhering to the wells were washed twice with 100 ml PBS. After this, 40–200 ml of freshly made neutral red (0.1% in distilled water diluted 1:10 with PBS, pH 7.4, just before use) was added to each well, and the plates were incubated for 90 min at 377C. After this incubation, the neutral red was removed and the wells of cells were gently washed two or three times with 100 ml 377C PBS. After removing the PBS, the dye was extracted from the remaining cells by adding 100 ml of 0.05 M citrate buffer plus 50% ethanol to each well. The plates were then agitated on a plate mixer at room temperature for 20 min, and the plate was read at 550 nm in a microtiter plate reader (Molecular Devices, Menlo Park, CA). Neutral red retention was compared in cells exposed to OPs and those exposed to 1% ethanol vehicle. Cells exposed to a 1% sodium dodecyl sulfate solution were used as a positive control for cytotoxicity. Data analysis. Percentages of activity for each assay were calculated, and the mean { SEM was determined for n Å 3–6 assays of each concentration of OP. For concentration–response studies, the mean and SEM of concentrations of the test compounds that inhibited esterases between 10 and 90% (greater than three concentrations/compound) after incubations of 1 hr at 377C were then entered into a linear regression program (Prism; GraphPad Software, San Diego, CA) set to determine regressions using log concentrations of the OPs. This program analyzed the data for slope, y values (apparent IC50 values), confidence intervals, and regression coefficient. Estimation of the comparative capability of the OPs to inhibit NTE and AChE was determined by dividing the apparent NTE IC50 value by the apparent AChE IC50 value. For visual representation of species differences, data were plotted, using the same software, with point-to-point curves used to better identify the tested concentrations.

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RESULTS

Tables 1 and 2 show apparent IC50 values, 95% confidence intervals for the apparent IC50 values, and regression coefficients (r 2) for the concentration-related inhibition of AChE and NTE in human and mouse neuroblastoma cells, respectively, following 1-hr incubations at 377C. The comparative ability of an OP to inhibit NTE or AChE was determined by dividing the apparent NTE IC50 value by the apparent AChE IC50 value. These ratios were very high (ú40) for OPs that do not cause OPIDN (paraoxon and malaoxon) and OPs unlikely to cause OPIDN (chlorpyrifos-oxon, dichlorvos, and trichlorfon). The ratios were considerably lower, and sometimes even less than 1, for OPs recognized for their capability to cause OPIDN (DFP, TSP, PSP, mipafox, DBVP, and DOVP). Although the apparent IC50 values for the various OPs differed between species, the ratios of apparent NTE IC50 value to apparent AChE IC50 value were high for paraoxon, malaoxon, chlorpyrifos-oxon, dichlorvos, and trichlorfon and low for DFP, TSP, PSP, mipafox, DBVP, and DOVP in both the human and the mouse neuroblastoma cell lines. Inhibition of esterases always occurred at OP concentrations lower than those necessary to cause cytotoxicity, as indicated by loss of capability to retain neutral red dye (Tables 1 and 2). Data on species differences between the OP-induced inhibition in human and murine cells and additional data on the relative inhibition of AChE and NTE in neuroblastoma cells exposed to OPs are provided in Fig. 1, which depicts semilogarithmic concentration response plots for OPs more likely to cause acute rather than delayed neurotoxicity (paraoxon, malaoxon, chlorpyrifos-oxon, dichlorovos, and trichlorfon), and in Fig. 2, which depicts concentration response plots for neuropathic OPs (DFP, TSP, PSP, mipafox, DBVP, and DOVP). Although species differences are evident in Fig. 1, overlap of the esterase concentration–response curves is not evident for either human or mouse cells, as AChE inhibition is õ20% of control at concentrations lower than the concentrations at which the NTE inhibition is ú80% of control. Species differences are also evident in Fig. 2, which shows that concentration–response curves for neuropathic OPs overlap considerably for OP-induced inhibitions of AChE and NTE. Figure 1, which includes the OPs more likely to cause acute cholinergic crisis (AChE inhibition) than OPIDN, indicates that species differences were greater for paraoxon and chlorpyrifos-oxon than for malaoxon, dichlorvos, or trichlorfon. For OPs noted for their ability to cause OPIDN (Fig. 2), the difference in esterase sensitivities between human and mouse cells was greater with DBVP than with DFP, TSP, PSP, mipafox, or DOVP. DISCUSSION

The results presented here demonstrate that neuroblastoma cells can be used to identify comparative differences in OP-

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induced inhibitions of AChE and NTE. The data show that cell lines of either human or murine origin could distinguish OPs more likely to cause acute cholinergic crisis (AChE inhibition) than OPIDN for 11 of the 11 test compounds included in the evaluation. The esterase inhibitions occurred at concentrations lower than those causing cytotoxicity, suggesting that this in vitro model could be used to examine cellular events that occur after esterases are inhibited. The apparent IC50 values determined in the present studies were often similar to the fixed-time IC50 values obtained during previous investigations that used the comparative inhibitions of NTE and AChE in brain homogenates from hens (the animal model for OPIDN) and humans to identify OPs likely to cause OPIDN (Lotti and Johnson, 1978; Richardson et al., 1993). This was true, even though conditions were somewhat different in studies with brain homogenates (e.g., 20 min incubation, AChE incubations at temperatures less than 377C, and multiple substrate concentrations). Table 3 provides a summary of fixed-time IC50 values in neuroblastoma cells and in tissue homogenates. The present study also reports that inhibition of the AChE and NTE activities found in SH-SY5Y and NB41A3 neuroblastoma cell lines by the OP test compounds used in this study reflects their in vivo toxicity (Johnson, 1982; Richardson, 1995; Ehrich et al., 1995a; Ecobichon, 1991). These cell lines, like tissue homogenates (Johnson, 1988; Richardson, 1995), however, are unlikely to have their esterase activities inhibited by protoxicants (Ehrich et al., 1994; Veronesi et al., 1997). Adequate metabolic activation is a common problem with in vitro esterase assay systems, whether they be neuronal cells or tissue homogenates (Chambers, 1992; Veronesi et al., 1997). Exogenous systems for biotransformation, although they could be somewhat difficult to incorporate, have been used with other systems for neurotoxicological investigations (Kohn and Dunham, 1993; Chow et al., 1986) and should be incorporated when using a cell culture system to identify protoxicants. The concentration–response studies reported here indicated that there are some species differences in sensitivities of living cells to OPs. Previous studies indicated that there were also species differences in basal quantities of AChE and NTE between human and murine cell lines (Veronesi and Ehrich, 1993a,b; Ehrich et al., 1995b; Veronesi et al., 1997). In the present study, both AChE and NTE activities of human cells were generally more susceptible to OP-induced inhibition than AChE and NTE activities of mouse cells. The apparent IC50 values in human cells, for example, were less than 13 of the apparent IC50 values in mouse cells for 7 of the 11 test compounds when AChE activity was determined and for 5 of the 11 test compounds when NTE was determined. Similarly in animal models, hen esterases have been demonstrated to be more sensitive to OP-induced inhi-

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TABLE 1 Apparent IC50 Values for Organophosphorus Compounds (OPs) Inhibiting Esterases and Causing Loss of Neutral Red Dye Retention in SH-SY5Y Human Neuroblastoma Cells a

OP Paraoxon

Malaoxon

Chlorpyrifos-oxon

Dichlorvos

Trichlorfon

DFP

TSP

PSP

Mipafox

DBVP

DOVP

AChE (Confidence interval) [r 2]

NTE (Confidence interval) [r 2]

Ratio of NTE IC50 to AChE IC50

Neutral red at 2–4 hr b [r 2]

0.0023 mM (0.00007, 0.03 mM) [0.92] 0.0097 mM (0.0095, 0.062 mM) [0.96] 0.00034 mM (0.000009, 0.005 mM) [0.96] 0.35 mM (0.083, 1.4 mM) [0.96] 1.2 mM (0.2, 4.8 mM) [0.96] 0.19 mM (0.041, 0.57 mM) [0.98] 0.028 mM (0.005, 0.12 mM) [0.96] 0.26 mM (0.11, 0.54 mM) [0.98] 13 mM (0.89, 18 mM) [0.91] 0.00094 mM (0.000003, 0.29 mM) [0.88] 0.72 mM (0.26, 4.5 mM) [0.98]

1600 mM (22, 10000 mM) [0.86] 740 mM (13, 1300 mM) [0.70] 0.069 mM (0.004, 0.58 mM) [0.95] 20 mM (3.6, 73 mM) [0.93] 82 mM (19, 250 mM) [0.92] 1.0 mM (0.29, 3.8 mM) [0.97] 0.043 mM (0.012, 0.13 mM) [0.95] 0.030 mM (0.028, 0.18 mM) [1.00] 14 mM (0.99, 81 mM) [0.90] 0.00076 mM (0.00007, 0.05 mM) [0.92] 0.40 mM (0.056, 2.9 mM) [0.95]

700,000

2600 mM [0.99]

76,000

3600 mM [0.99]

200

1900 mM [0.72]

42

1600 mM [0.89]

68

4100 mM [0.91]

5.3

No effect

1.5

110 mM [0.94]

0.12

43 mM [0.98]

1.1

No effect

0.81

520 mM [0.94]

0.56

380 mM [0.99]

a Esterase inhibitions (acetylcholinesterase, AChE; neuropathy target esterase, NTE) were determined following 1 hr of incubation (377C) with the OPs at concentrations up to 1003 M. Concentrations inhibiting esterase activities by 50% at that time and temperature (apparent IC50 values) were calculated from semilogarithmic curves derived from at least three assays that included at least three concentrations giving between 10 and 90% inhibition of activity (Prism; GraphPad, San Diego, CA). The apparent IC50 values are followed by their 95% confidence intervals (given in parentheses) and by r 2 for the composite inhibition line. Esterase activities in untreated SH-SY5Y cells (mean { SD, n Å 20–28) were AChE Å 76.4 { 10.2 nmol/min/mg protein and NTE Å 22.7 { 2.3 nmol/min/mg protein. b Neutral red retention was determined following incubation of cells with OP for 1–72 hr at 377C, with the exception of PSP, mipafox, and DFP (earliest determination at 4 hr). Only the 2-hr results (or 4-hr results for PSP, mipafox, and DFP) are included in the table above. Calculation of the percentage of dye retention was based on absorbance { SEM of five to eight incubations on different days of assay, with six wells run per assay day. Untreated cells, 0.229 { 0.023 absorbance units (n Å 31, range Å 0.078–0.567) for incubation periods of 1 to 72 hr. Prism (GraphPad) allowed for calculation of the apparent IC50 value if three points were on a regression line even though cells were not incubated with concentrations of OP ú 1000 mM. For mipafox and DFP, no loss of dye retention was seen after 4 hr of incubation, even at 1000 mM This is indicated by ‘‘no effect.’’

bition relative to esterases of rats (Ehrich et al., 1995a). The reasons for this species difference are as yet undetermined, although differences in enzymes involved in detoxification of OPs (e.g., carboxylesterases and A-esterases) may be contributing factors (Veronesi et al., 1993a; Ehrich et al., 1995b;

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Maxwell, 1992). In the present study, carboxylesterases of the human cell line appear more sensitive to OP-induced inhibition than carboxylesterases of the mouse cell line (apparent IC50 values in human cells were less than 13 of the apparent IC50 values in mouse cells for 8 of the 11 test

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TABLE 2 Apparent IC50 Values for Organophosphorus Compounds (OPs) Inhibiting Esterases and Causing Loss of Neutral Red Dye Retention in NB41A3 Murine Neuroblastoma Cells a

OP Paraoxon

Malaoxon

Chlorpyrifos-oxon

Dichlorvos

Trichlorfon

DFP

TSP

PSP

Mipafox

DBVP

DOVP

AChE (Confidence interval) [r 2]

NTE (Confidence interval) [r 2]

0.047 mM (0.0036, 0.34 mM) [0.97] 0.047 mM (0.00008, 0.67 mM) [0.89] 0.011 mM (0.0014, 0.056 mM) [0.97] 0.55 mM (0.0031, 11 mM) [0.87] 1.4 mM (0.64, 2.9 mM) [0.99] 0.056 mM (0.0022, 0.58 mM) [0.95] 0.11 mM (0.0003, 3.2 mM) [0.94] 0.085 mM (0.016, 0.34 mM) [0.98] 46 mM (4.9, 210 mM) [0.91] 0.010 mM (0.0006, 0.27 mM) [0.94] 6.4 mM (1.4, 29 mM) [0.97]

80 mM (14, 28 mM) [0.93] 1400 mM (740, 1400 mM) [0.69] 0.96 mM (0.054, 1.9 mM) [0.82] 82 mM (6.1, 440 mM) [0.91] 140 mM (35, 450 mM) [0.98] 0.30 mM (0.027, 1.8 mM) [0.96] 0.0076 mM (0.001, 0.040 mM) [0.99] 0.033 mM (0.00013, 0.97 mM) [0.84] 120 mM (9.4, 600 mM) [0.87] 0.096 mM (0.008, 1.1 mM) [0.89] 2.0 mM (0.53, 7.7 mM) [0.97]

Ratio of NTE IC50 to AChE IC50

Neutral red at 2–4 hr b [r 2]

1700

360 mM [0.68]

30,000

4000 mM [0.44]

87

80 mM [0.98]

150

980 mM [0.68]

100

2500 mM [0.68]

5.4

4200 mM [0.88]

0.07

78 mM [0.93]

0.39

2500 mM [0.99]

2.6

No effect

9.6

440 mM [0.83]

0.31

820 mM [0.92]

a Esterase inhibitions (acetylcholinesterase, AChE; neuropathy target esterase, NTE) were determined following one hr of incubation (377C) with the OPs at concentrations up to 1003 M. Apparent IC50 values were calculated from semilogarithmic curves derived from at least three assays that included at least three concentrations, giving between 10 and 90% inhibition at this time and temperature. The apparent IC50 values are followed by their 95% confidence intervals (given in parentheses) and by r 2 for the composite inhibition line. Esterase activities in untreated NB41A3 cells, mean { SD, n Å 23–38) were AChE Å 10.3 { 1.1 nmol/min/mg protein and NTE Å 3.37 { 0.22 nmol/min/mg protein. b Neutral red retention was determined following incubation of cells with OP for 1–72 hr, with the 2-hr value reported above. Prism (GraphPad) allowed for calculation of the apparent IC50 value if three points were on a regression line even though cells were not incubated with concentrations of OP ú 1000 mM. For mipafox, no loss of dye retention was seen, even at 1000 mM. This is indicated by ‘‘no effect.’’ Control values, 0.319–0.405 absorbance units or 3.31 { 0.14 mg/ml (100% { 4, n Å 6).

compounds used for the present study) (M. Ehrich, unpublished data). Nervous tissue homogenates, although relatively simple to use for in vitro inhibition studies, differ from the in vitro model described here in several ways. Tissue homogenates obviously require the sacrifice of animals, thereby preventing study of post-esterase inhibition changes in living tissue. Although primary cultures of pure neurons might also be

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used to examine OP inhibition, their cost and technical requirements are considerably greater than those necessary for cell lines (Freshney, 1987; Conn, 1990; Funk et al., 1994). A compelling advantage for using cell lines to address OP inhibition is that cells of human origin can be readily obtained (American Type Culture Collection, Rockville, MD; Veronesi and Ehrich, 1993a), and it is all but impossible for most laboratories to obtain human test material for primary

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FIG. 1. Concentration–response curves for inhibition of acetylcholinesterase (AChE) and neuropathy target esterase (NTE) in neuroblastoma cell lines of human and murine origin by organophosphorus compounds (OPs) more likely to cause acute cholinergic crisis (AChE inhibition) than organophosphorus-induced delayed neuropathy (OPIDN). (a) Paraoxon, (b) malaoxon, (c) chorpyrifos-oxon, (d) dichlorvos (DDVP), (e) trichlorfon. Point-to-point composite curves are provided to aid visualization (Prism; GraphPad, San Diego, CA). Each curve represents at least three different assays that included at least three concentrations of OPs that provided values between 10 and 90% of values in vehicle-treated cells.

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FIG. 2. Concentration–response curves for inhibition of AChE and NTE in neuroblastoma cells exposed to OPs capable of inducing OPIDN. (a) DFP, (b) TSP, (c) PSP, (d) mipafox, (e) DBVP, (f) DOVP. See legend to Fig. 1 for explanation.

cultures or tissue homogenates. Neuronal cell lines from the hen, the currently accepted animal model for OP studies, however, are not currently available from commercial sources. The present model does not intend to substitute for the currently accepted hen test (US EPA, 1991), which

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includes esterase inhibition, clinical signs, and neuropathology as endpoints, but does suggest that cell models be considered to act as an adjunct test in view of their cost, human availability, and accuracy in identifying OPIDN-causing OPs. The present data adds to previous studies using other

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TABLE 3 Comparison of Apparent IC50 Values in Neuroblastoma Cells with Fixed-Time IC50 Values in Tissue Homogenates Exposed to OPs a Apparent IC50 values in neuroblastoma cells

OP DFP

AChE NTE AChE NTE AChE NTE AChE NTE

PSP Chlorpyrifosoxon Mipafox

Å Å Å Å Å Å Å Å

0.2–0.6 mM 0.3–1.0 mM 0.1–0.3 mM 0.03 mM 0.003–0.1 mM 0.07–1.0 mM 13–46 mM 4–120 mM

Fixed-time IC50 values in tissue homogenates AChE NTE AChE NTE AChE NTE AChE NTE

Å Å Å Å Å Å Å Å

0.6–0.8 mM 0.7–1.2 mM 0.1–0.3 mM 0.002–0.003 mM 0.002 mM 0.2 mM 6–41 mM 2–13 mM

Paraoxon

AChE Å 0.002–0.05 mM

AChE Å 0.005–0.01 mM

DBVP

NTE Å 0.0007–0.096 mM

NTE Å 0.002 mM

References Lotti and Johnson, 1978 Lotti and Johnson, 1978 Richardson et al., 1993 Lotti and Johnson, 1978; Novak and Padilla, 1986; Correll and Ehrich, 1987; MacKay et al., 1996 Lotti and Johnson, 1978; Correll and Ehrich, 1987 Moretto et al., 1989

Apparent IC50 values in neuroblastoma cells of human and murine origin were obtained following 1 hr of incubation at 377C. Fixed-time AChE inhibitions in brain homogenates from humans, rats, and/or hens were obtained following 5- to 20-min incubations either at or converted to 377C. Fixedtime NTE inhibitions in brain homogenates were obtained following 10- to 30-min incubations at 377C. Chlorpyrifos-oxon values were obtained using several concentrations of acetylthiocholine (for AChE) and phenyl valerate (for NTE). a

in vitro systems that suggested, if judiciously selected and characterized, these systems could eventually be used to screen esterase inhibitors (Dudek and Richardson, 1982, Bertoncin et al., 1985; Fedalei and Nardone, 1983; Carrington et al., 1985; Knoth Anderson et al., 1992; Nostrandt and Ehrich, 1992; Ehrich et al., 1994; Veronesi et al., 1993a,b; 1996; Funk et al., 1994). In summary, the present data show that the relative inhibition of NTE and AChE in neuroblastoma cells can distinguish OPs more likely to cause cholinergic crisis from neuropathic OPs. This provides possibility for an in vitro alternative to in vivo models currently used (US EPA, 1991) for screening of these potential neurotoxicants. ACKNOWLEDGMENTS The technical support of Kent Carlson, Kristel Fuhrman, and June Mullins is gratefully acknowledged. This was supported by the U.S. EPA Neurotoxicology Division (CR 821928-01-0). The work in the manuscript has been reviewed by the Health Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for submission. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

REFERENCES Bertoncin, D., Russolo, A., Carold, S., and Lotti, M. (1985). Neuropathy target esterase in human lymphocytes. Arch. Environ. Health 40, 139– 144. Carrington, C. D., Carringon, M. N., and Abou-Donia, M. B. (1985). Neurotoxic esterase in cultured cells: An in vitro alternative for the study of

AID

FAAT 2330

/

6k1e$$$$23

07-30-97 13:28:34

organophosphorus compound-induced delayed neurotoxicity. In Alternative Methods in Toxicology, In Vitro Toxicology (A. M. Goldberg, Ed.), Vol. 3, pp. 455–465. Mary Ann Liebert Inc., New York. Chambers, J. E. (1992). The role of target-site activation of phosphorothionates in acute toxicity. In Organophosphates, Chemistry, Fate, and Effects (J. E. Chambers and P. E. Levi, Eds.), pp. 229–239. Academic Press, San Diego. Chambers, J. E., and Levi, P. E. (1992). Organophosphates, Chemistry, Fate and Effects. Academic Press, San Diego. Chow, E., Seiber, J. N., and Wilson, B. W. (1986). Isofenphos and an in vitro activation assay for delayed neuropathic potential. Toxicol. Appl. Pharmacol. 83, 178–183. Conn, P. M. (1990). Cell Culture, Methods in Neurosciences, Vol. 2. Academic Press, San Diego. Correll, L., and Ehrich, M. (1987). Comparative sensitivities of avian neural esterases to in vitro inhibition by organophosphorus compounds. Toxicol. Lett. 36, 197–204. Correll, L., and Ehrich, M. (1991). A microassay method for neurotoxic esterase determinations. Fundam. Appl. Toxicol. 16, 110–116. Dudek, B. R., and Richardson, R. J. (1982). Evidence for the existence of neurotoxic esterase in neural and lymphatic tissue of the adult hen. Biochem. Pharmacol. 31, 1117–1121. Ecobichon, D. J. (1991). Toxic effects of pesticides. In Casarett and Doull’s Toxicology, the Basic Science of Poisons (M. O. Amdur, J. Doull, and C. D. Klaassen, Eds.), 4th ed., pp. 565–622. Pergamon, New York. Ehrich, M., Correll, L., and Veronesi, B. (1994). Neuropathy target esterase inhibition by organophosphorus esters in human neuroblastoma cells. NeuroToxicology 15, 309–314. Ehrich, M., Jortner, B. S., and Padilla, S. (1995a). Comparison of the relative inhibition of acetylcholinesterase and neuropathy target esterase in rats and hens given cholinesterase inhibitors. Fundam. Appl. Toxicol. 24, 94–101.

ftoxas

OPs AND ESTERASE INHIBITION IN NEUROBLASTOMA CELLS Ehrich, M., Correll, L., Carlson, K., Wilcke, J., and Veronesi, B. (1995b). Examination of culture conditions on esterase activities in human and mouse neuroblastoma cells. In Vitro Toxicol. 8, 199–207. Fedalei, A., and Nardone, R. M. (1983). An in vitro alternative for testing the effect of organophosphates on neurotoxic esterase activity. In Alternative Methods in Toxicology, Product Safety Evaluation (A. Goldberg, Ed.), Vol. 1, pp. 253–269. Mary Ann Liebert Inc., New York. Freshney, R. I. (1987). Culture of Animal Cells, a Manual of Basic Techniques, 2nd Ed. Wiley–Liss, New York. Funk, K. A., Liu, C. H., Wilson, B. W., and Higgins, R. J. (1994). Avian embryonic brain reaggregate culture system. I. Characterization for organophosphorus compound toxicity studies. Toxicol. Appl. Pharmacol. 124, 149–158. Hollinghaus, J. G. (1984). Chemistry and metabolism of delayed neurotoxic organophosphorus esters. In Proceedings of the Fourteenth Conference on Environmental Toxicology, pp. 76–105. [AFAMRL-TR-83-099 from NTIS, Springfield, VA]. 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. (1982). The target for initiation of delayed neurotoxicity by organophosphorus esters: Biochemical studies and toxicological applications. In Reviews in Biochemical Toxicology (E. Hodgson, J. R. Bend, and R. M. Philpot, Eds.), Vol. 4, pp. 141–212. Elsevier Sci. Pub., New York. Johnson, M. K. (1988). Sensitivity and selectivity of compounds interacting with neuropathy target esterase. Biochem. Pharmacol. 37, 4095–4104. Knoth Anderson, J., Veronesi, B., Jones, K., Lapadula, D. M., and AbouDonia, M. B. (1992). Triphenyl phosphite-induced ultrastructural changes in bovine adrenomedullary chromaffin cells. Toxicol. Appl. Pharmacol. 112, 110–119. Kohn, J., and Durham, H. D. (1993). S9 liver fraction is cytotoxic to neurons in dissociated culture. NeuroToxicology 14, 381–386. Lotti, M., and Johnson, M. K. (1978). Neurotoxicity of organophosphorus pesticides: Predictions can be based on in vitro studies with hen and human enzymes. Arch. Toxicol. 41, 215–221. MacKay, C. E., Hammock, B. D., and Wilson, B. W. (1996). Identification and isolation of a 155-kDa protein with neuropathy target esterase activity. Fundam. Appl. Toxicol. 30, 23–30.

AID

FAAT 2330

/

6k1e$$$$23

07-30-97 13:28:34

63

Marrs, T. C. (1993). Organophosphate poisoning. Pharmacol. Ther. 58, 51– 66. Maxwell, D. M. (1992). Detoxication of organophosphorus compounds by carboxylesterase. In Organophosphates, Chemistry, Fate, and Effects (J. E. Chambers and P. E. Levi, Eds.), pp. 183–199. Academic Press, San Diego. Moretto, A., Lotti, M., and Spencer, P. S. (1989). In vivo and in vitro regional differential sensitivity of neuropathy target esterase to di-n-butyl2,2-dichlorovinyl phosphate. Arch. Toxicol. 63, 469–473. Nostrandt, A. C., and Ehrich, M. (1992). Development of a model cell culture system in which to study early effects of neuropathy-inducing organophosphorus esters. Toxicol. Lett. 60, 107–114. Nostrandt, A. C., and Ehrich, M. (1993). Modification of mipafox-induced inhibition of neuropathy target esterase in neuroblastoma cells of human origin. Toxicol. Appl. Pharmacol. 121, 36–42. Novak, R., and Padilla, S. (1986). An in vitro comparison of rat and chicken brain neurotoxic esterase. Fundam. Appl. Toxicol. 6, 464–471. Richardson, R. J. (1995). Assessment of the neurotoxic potential of chlorpyrifos relative to other organophosphorus compounds: A critical review of the literature. J. Toxicol. Environ. Health 44, 135–165. Richardson, R. J., Moore, T. B., Kayyall, U. S., Fowke, J. H., and Randall, J. C. (1993). Inhibition of hen brain acetylcholinesterase and neurotoxic esterase by chlorpyrifos in vivo and kinetics of inhibition by chlorpyrifosoxon in vitro: Application to assessment of neuropathic risk. Fundam. Appl. Toxicol. 20, 273–279. U.S. Environmental Protection Agency (1991). Pesticide Assessment Guidelines, Subdivision E. Hazard Evaluation: Human and Domestic Animals. [Addendum 10: Neurotoxicity, series 81, 82, and 83. Office of Prevention, Pesticides and Toxic Substances, Washington, DC. EPA 540/09-1-123. Available from NTIS, Springfield, VA, PB91-154617] Veronesi, B., and Ehrich, M. (1993a). Differential cytotoxic sensitivity in mouse and human cell lines exposed to organophosphate insecticides. Toxicol. Appl. Pharmacol. 120, 240–246. Veronesi, B., and Ehrich, M. (1993b). Using neuroblastoma cell lines to examine organophosphate neurotoxicity. In Vitro Toxicol. 6, 57–65. Veronesi, B., Padilla, S., Blackmon, K., and Pope, C. (1991). Murine susceptibility to organophosphorus-induced delayed neuropathy (OPIDN). Toxicol. Appl. Pharmacol. 107, 311–324. Veronesi, B., Ehrich, M., Blusztajn, J. K., Oortgiessen, M., and Durham, H. D. (1997). Cell culture models of interspecies selectivity to organophosphorus insecticides. NeuroToxicology 18, 283–298.

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