Toxic. in Vitro Vol. 6, No. 3, pp. 261-266, 1992 Printed in Great Britain
0887-2333/92 $5.00 + 0.00 Pergamon Press Ltd
ANTICHOLINESTERASE ACTIVITY OF ORGANOPHOSPHATE NERVE AGENTS IN N E U R O N A L TISSUE C U L T U R E T. W. SAWYER*, M. T. WEISSand R. J. UNGER Biomedical Defence Section, Defence Research Establishment Sutiield, Box 4000, Medicine Hat, Alberta, Canada T1A 8K6 (Received 26 August 1991; revisions received 30 October 1991)
Akstract--Primary chick embryo forebrain culture was examined with respect to its usefulnessas a model to study organophosphate (OP) anticholinesterases. The acetylcholinesterase (ACHE) activity of these cultures increased with time in culture and paralleled the development in ovo of this enzyme.The neuronal cells were extremely sensitive to the anticholinesterase effects of OP nerve agents, and experiments with soman indicated that enzyme inhibition was rapid and persistent. The potencies of several OP nerve agents as inhibitors of AChE activity in vitro paralleled their literature-reported toxicities in vivo.
The defence community is interested in the organophosphate (OP) nerve agents, owing to their use as chemical warfare agents. These compounds function by inhibiting the activity of plasma cholinesterase (pseudocholinesterase), as well as acetylcholinesterase (ACHE) in red blood cells and neural tissue. This results in the accumulation of acetylcholine at neuroreceptor transmission sites and a subsequent massive overstimulation of the cholinergic system (Minton and Murray, 1988). To study this class of compounds, a mouse embryo neuronal culture system, which exhibited extreme sensitivity to the anticholinesterase effects of these agents in a manner that paralleled their toxicities in vivo, has previously been described (Sawyer et al., 1991). This in vitro system, although used to advantage, was found to be impractical for the cultivation and maintenance of large numbers of cultures. This was due to problems including (1) relatively high cost (but still substantially less than comparable studies in vivo), (2) labour intensiveness, (3) fragility to treatment manipulation, and (4) difficulties in obtaining correctly-staged pregnant dams at Defence Research Establishment Suffield facilities. A search was thus initiated for a replacement for this system that would retain the
desirable characteristics of the mouse neuron system, but would not present the difficulties outlined above. During this search, purified AChE preparations and neuronal cell lines were not considered as suitable candidates. Although these models have been demonstrated to be sensitive to the anticholinesterase activity of carbamates and OPs (Iyaniwura, 1989; Segal and Fedoroff, 1989a,b), it was felt that primary tissue cultures would be a more appropriate model with which to pursue future studies to examine the efficacies of various drug regimens against poisoning by OP nerve agents. This report describes the characteristics of a chick embryo forebrain neuron culture system that has some utility as a tool with which to study OP nerve agents.
MATERIALS AND METHODS
Chemicals. Tabun (GA, ethyl N,N-dimethyl phosphoramidocyanidate), sarin (GB, isopropyl methylphosphonofluoridate), soman (GD, pinacolyl methylphosphonofluoridate), cyclohexyl methylphosphonofluoridate (GF) and ethyl S-2-diisopropyl aminoethyl methylphosphorothiolate (VX) were synthesized by the Chemical Biological Defence Section, Defence Research Establishment Suffield. Diisopropyl fluorophosphate (DFP), as well as all bio*To whom correspondence should be addressed. Abbreviations: AChE = acetylcholinesterase;DIV = days in chemicals, were purchased from Sigma Chemical vitro; DFP = diisopropyl fluorophosphate; GA = tabun, Company (St Louis, MO, USA). Horse serum (HS) ethyl N,N-dimethyl phosphoramidocyanidate; GB = and 35 mm Lux culture dishes were acquired from sarin, isopropyl methylphosphonofluoridate; G D = Flow Laboratories (Mississauga, Ontario, Canada), soman, pinacolyl metbylphosphonofluoridate; GF = cyclohexyl methylphosphonofluoridate; HS = horse while a modified minimum essential medium serum; ICs0= median inhibitory concentration; (mMEM) was prepared as described by Hertz et al. mMEM = modified minimum essential medium; (1982). Tritiated acetylcholine chloride was obtained OP = organophosphate; PBS = phosphate buffered saline; VX = ethyl S-2-diisopropyl aminoethyl from Amersham Corporation (Oakville, Ontario, Canada). methylphosphorothiolate.
T.W. SAWYERet al.
C h i c k e m b r y o neuron culture. The methodology for the culturing of primary chick embryo forebrain neurons was based largely on previously described techniques (Pettmann et al., 1979; Sensenbrenner et al., 1978). Fertile Mountain Hubbard chicken eggs were obtained from a commercial hatchery. The eggs were incubated for 8 days, and then the embryos were aseptically removed and the lobes of the telencephalon isolated. After removal of the meninges, the lobes were triturated with a pasteur pipette and then trypsinized (0.25%) with gentle agitation for 2 min. The trypsin was deactivated by the addition of medium supplemented with HS (20%). The resulting cell suspension was triturated once more with a pasteur pipette and centrifuged. The cell pellet was resuspended in cold m M E M containing 30 mM-glucose, filtered through Nitex mesh (70/~m pore size), and then counted and seeded at 2 x 10 6 cells per 2.0 ml medium per 35-mm Lux culture dish. These culture dishes had previously been coated with approx. 1.5ml L-polylysine (12.5/~g/ml) to facilitate neuronal attachment (Letourneau, 1975; Yavin and Yavin, 1974), incubated overnight and then rinsed twice with sterile phosphate buffered saline (PBS) before cell seeding. After the cultures had been incubated at 37°C for 30 min to allow the neurons to attach, the medium containing unattached cells (mostly non-neuronal) was removed and warm (37°C) m M E M containing 5% HS and 30mMglucose supplemented with penicillin (10 units/ml), streptomycin (10 #g/ml) and Fungizone (0.25 #g/ml) (Gibco Laboratories, Grand Island, NY, USA) was gently added. The cultures were then returned to an incubator containing a humidified atmosphere of 95/5% (v/v) air/CO2 and left undisturbed until used for experimentation. C h e m i c a l treatments. Neuron cultures were routinely treated with OP at 8 days in vitro (DIV). On the day of agent exposure, the spent medium was carefully aspirated off and 2 ml fresh medium was gently added. The test compound was diluted in potassium phosphate buffer, pH 6.0, so that the desired final test compound concentration could be reached by adding 0.1 ml of the test agent stock solution to each plate. The cultures were exposed to the agents at log interval doses for 30 min at room temperature, and then harvested by scraping. The cell suspensions were spun down at 1000g for 3-5min and the pellet resuspended in 0.1 ml potassium phosphate buffer, pH 7.0. Time course studies. To examine the development of AChE in vitro, chick embryo cultures were harvested at various time points starting on the day of seeding (0 DIV) and out to 14 DIV. To investigate the development of AChE in ovo, embryos were isolated at various times of incubation. One time point was taken 8 days after hatching. The forebrains were isolated and homogenized in nine volumes of potassium phosphate buffer, pH 7.0.
The inhibition of AChE was examined by incubating neuron cultures (8 DIV) with a maximally inhibiting dose of soman (10 -8 M) for predetermined time periods. At the end of the time period the treatment medium was quickly aspirated off and 2 ml ice-cold potassium phosphate buffer, pH 7.0, was added. The cultures were then harvested as described above. Regeneration of inhibited AChE was studied by incubating cultures (5 DIV) with 10 -8 M-soman for 30 min. The treatment medium was then aspirated off and the cultures were gently rinsed with 2 ml warm (37°C) PBS. The cultures were then re-fed with a 50/50 (v/v) mixture of fresh medium and conditioned medium from 5-day-old cultures, that had been supplemented with cycloheximide (2/~ g/ml). Control cultures received buffer only and were re-fed as described above, with or without cycloheximide. Samples were harvested immediately after rinsing and for 4 consecutive days thereafter. Biochemistry. Aliquots of the whole cell suspension were taken for protein and AChE determinations. Protein determinations were carried out using Pierce Protein Assay Reagent (Pierce Chemical Company, Rockford, IL, USA) with bovine serum albumin as the protein standard. Acetylcholinesterase activity was measured using the method of Johnson and Russell (1975). Median inhibitory concentrations (IC50) were determined graphically. RESULTS
Time course studies
The morphological development of the neuron cultures paralleled previous observations of the development of pure cultures of chick embryo neurons (Sensenbrenner et al., 1978). Within 5-15 min after seeding, the spherical cells were anchored and evenly distributed on the bottom of the culture vessel, and by 3 hr in vitro the cells were starting to flatten out, with small processes just visible under high magnification. After 24 hr in vitro, these neurite extensions were easily discerned, and the neurons could be seen to be migrating into aggregates. At 3 DIV the aggregates were interconnected by an extensive latticework of neurite processes, and by 6-8 DIV the aggregation appeared essentially complete. Beyond 9 DIV the aggregates appeared to degenerate slowly so that by 12-14 DIV few aggregates were attached to the substratum. Aggregates that were still anchored at this point possessed few, if any, processes. Figure 1 shows the total protein per culture dish, as well as the AChE specific activity as a function of time in culture. After an initial decrease in total protein per dish on day 1, the protein values increased to a maximum by 6-7 DIV. Thereafter the protein gradually decreased so that by 14 DIV the total protein per dish had returned to starting values. The AChE specific activity gradually increased with time in culture out to 13 DIV (251.9 46.3nmol/mg protein/min, n = 3 experiments) when it appeared
OP nerve agents in neuronal cultures
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GD i n c u b a t i o n
in v i t r o
Fig. 1. Time course of the development of protein and AChE in vitro. Cultures were seeded on day zero and harvested at selected time points to determine total protein/culture dish ( I ) and AChE specific activity (A). Each point represents the mean + standard deviation of three experiments utilizing two plates per time point.
Fig. 3. Acetylcholinesterase inhibition as a function of GD incubation time. Cultures were treated at 8 DIV for varying periods with GD (10 -s M) and then the treatment medium was aspirated off, ice-cold potassium phosphate buffer was added immediately and the cultures were harvested. Values represent the mean + standard deviation of two experiments utilizing three replicates per time point.
to plateau. The A C h E specific activity of chick forebrain (Fig. 2) increased throughout the time period measured in o v o (20 days in o v o : 348.2 + 66.7 nmol/mg protein/min, n = 4 animals) and was unchanged 8 days after hatching. Figure 3 shows the inhibition of A C h E by G D as a function of time. Inhibition was rapid: it was first apparent by 15 sec and became over 50% complete by 1 min of incubation with GD. Thereafter the A C h E activity gradually decreased further, approaching zero values with time. Figure 4 shows the effects on A C h E levels after 30 min G D incubation, compared with untreated controls. In those cultures not treated with G D or cycloheximide, A C h E specific activity gradually increased over the 4-day test period. This was in contrast to those cultures that had been treated with cycloheximide only and in which A C h E activities persisted at similar values throughout the test period. In cultures treated with G D and then re-fed with medium supplemented with cycloheximide post-treatment (Fig. 4), A C h E activity decreased to approx. 1% of control values but then regained about one-third of the original activity 2
days after treatment, persisting at these levels for a further 2 days. Pretreatment of the cultures with cycloheximide 2 days before G D treatment did not change these observations markedly (data not shown). Figure 5 and Table 1 show the potencies of several classical nerve agents in chick embryo neurons. Figure 5 illustrates the typical sigrnoid dose-response curve obtained with this class of compounds in this system. At low doses of OP the A C h E activity is not much changed from that of buffer-treated controls; however, at higher doses the slope of the curve increases sharply and then the A C h E activity gradually approaches zero asymptotically. Table 1 shows the relative potencies of these agents as a function of the dose required to inhibit 50% of the control culture A C h E specific activity (IC50). VX was the most potent anticholinesterase, followed by G D , GB, GF, GA, and then D F P in order of decreasing anticholinesterase activity.
E 3 E
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~ zo ~_ = 0
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Fig. 2. Time course of the development of AChE specific activity. Mountain Hubbard chicken eggs were harvested starting on day 8 of incubation. One time point was taken 8 days after hatching. Values represent the mean _ standard deviation of four animals at each time point.
Fig. 4. Effect of GD on AChE activity. Cultures were treated at 5 DIV for 30 min, rinsed once with PBS and then re-fed. Values represent the mean + standard deviation of two experiments utilizing three replicates per time point. Control cultures re-fed with normal medium (O); control cultures re-fed with medium supplemented with 2/~g cycloheximide/ml ( I ) ; cultures treated with 10-8 M-GD and re-fed with medium supplemented with 2#g cycloheximide/ml (A).
T.W. SAWYERet al.
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Log dose (pM) Fig. 5. Dose response of organophosphate nerve agents in chick embryo forebrain neuron cultures. Cultures were treated at 8-9 days in vitro with GA (O), GB (0), GD ( I ) , GF (&) and VX ( + ). The cultures were then incubated for 30 min at room temperature before harvesting. Whole-cell suspensions were assayed for acetylcholinesterase activity and protein content. Values represent the averages of three experiments utilizing three replicate cultures per dose. DISCUSSION
The development of AChE activity in the chick embryo cultures seemed to contradict the conclusions of an apparent decline in viability after 8-9 DIV, as evidenced by visual inspection and total protein (Fig. 1). The AChE specific activity continued to increase even after protein values had peaked, so that by 14 DIV the AChE specific activity had increased three-fold from the time of seeding. This closely paralleled our observations of a developmental increase in AChE in ovo. These findings have been well documented in other laboratories in both chick (Louis et al., 1981; Peterson et al., 1974; Werner et al., 1971) and rodent embryos (Seeds, 1971); however, support for our observations of an increase in AChE activity in vitro is not as clear. Although developmental increases in this enzyme have been reported previously in aggregate (Briichner, 1985; Honneger and Richelson, 1976; Peterson et al., 1974; Richelson, 1976; Seeds, 1971; Trapp and Richelson, 1980) and explant (Honneger and Richelson, 1976; Kim and
Tunnicliff, 1974; Schlapfer et al., 1972) cultures, the majority of attempts at detecting this increase in surface culture have been unsuccessful, regardless of tissue age or species (Honneger and Richelson, 1976; Schlapfer et al., 1972; Seeds, 1971; Shapiro and Schrier, 1973; Trapp and Richelson, 1980; Werner et al., 1971). Typically, in surface cultures of nervous tissue the AChE activity declines and never regains the levels observed at the time of plating. This has led to speculation that the multicell-type environment, as well as the high degree of three-dimensional organization present in vivo, and in explant and aggregate neural cultures, is an absolute requirement for normal AChE development (Briichner, 1985; Honneger and Richelson, 1976; Trapp and Richelson, 1980). Our data clearly contradict this, and closely agree with studies (Louis et al., 1981) that show a parallel development of AChE in vivo, and in surface cultures of primary chick embryo forebrain neurons. The reasons for our findings of a developmental increase in AChE in vitro are not clear, although they may reflect improvements in cell culture medium. The next phase of work investigated the characteristics of the inhibition of AChE activity in these cultures. A dose of soman was selected that would maximally inhibit ACHE, and the inhibition was examined as a function of time. The inhibition of chick embryo neuron AChE was shown to occur extremely quickly and to be essentially complete within minutes. Studies were next carried out to examine the persistence of this inhibition. Experiments (Fig. 4), utilizing the protein synthesis inhibitor cycloheximide immediately post-treatment, showed that although AChE inhibition was close to 100% immediately after 30rain 10-SM-GD incubation, approximately one-third of this activity was recovered within 2 days. This seems to be consistent with reports that suggest the existence of large reservoirs of AChE subunits that are resistant to OP inhibition (Rotundo, 1988). Interestingly, however, if protein synthesis was inhibited by cycloheximide for
Table 1. Anticholinesteraseactivitiesof organophosphatenerveagentsin chickembryoneuronculturescomparedwiththeir toxicities in mouseand rat lCso (M) (chick embryo LD~o(#g/kg) LDso (,ug/kg) Compound neuron ACHE) (mouse, sc) (rat, sc)* D F P (diisopropyl fluorophosphate) G A (ethyl N,N-dimethyl phosphoramidocyanidate) G B (isopropyl methylphosphonofluoridate) G D (pinacolyl methylphosphonofluoridate) G F (cyclohexyl methylphosphonofluoridate) V X (S-2-diisopropyl aminoethyl methylphosphorothiolate)
(1.51 + 1.23) x n= 3 (3,01 _+ 0.90) x n = 7 (3,t6_+0.80) x n = 8 (1,49 + 0.35) x n = I1 (6.48 ± 2.17) x n = 3 (6.20 _+ 1.64) × n=8
10 7 3000*
10 s 10 9 l0 9 10 9 10 to 21~"
*RTECS (1991). tSawyer et al. (1991). ~Sterri (1981). Linearregressionanalysisof ICs0v. LDs0(mouse)and IC50v. LDs0(rat) yieldedcorrelationcoetficientsof 0.996 and 0.987, respectively.
OP nerve agents in neuronal cultures the 2 days before G D treatment, the cultures still regained about 35% of their original activity by 2 days post-treatment. This may suggest the existence of a store of A C h E subunits that are not sensitive to OP inhibition and are mobilized only when cellular A C h E levels are depressed. In none of these studies did G D produce any visible morphological alteration. Studies were next conducted to ascertain whether the sensitivity of the chick embryo neuron cultures to OP nerve agents was dependent on their time in culture. Several time points were selected (2, 7, 8, 9 and 14 DIV) and the ICs0 of soman was determined. The ICs0 values ranged from 1.14× 10 9~ to 1.53 x 10 -9 M in these experiments and indicated that the high sensitivity of the neurons to the anticholinesterase effects of soman did not vary markedly with the age of the culture. On the basis of this study, as well as on the protein and A C h E time courses, neuron cultures were used at an age of 7-9 DIV for anticholinesterase studies. The final phase of these studies was carried out to determine the anticholinesterase activities of the well known OP nerve agents tabun, sarin, soman, G F , VX and DFP. The ICs0 values for these compounds illustrate their potent anticholinesterase activities and were very similar to those values previously obtained in mouse cortical neuron cultures (Sawyer et al., 1991). In addition, the rank order of potencies of these compounds as inhibitors of A C h E in vitro closely paralleled their acute toxicities in vivo. In summary, chick embryo forebrain neuron cultures appear to be a useful tool with which to predict the potential toxicity in vivo of OP nerve agents, and also to eliminate many of the problems presented by mouse embryo neuronal cultures. These cultures are economical, and relatively easy to initiate and maintain. In addition, their resiliency and approximately lO-day lifespan in vitro would permit their use in experimental protocols that examine the efficacy of prophylactic and therapeutic drug regimens to protect against poisoning by OP nerve agents.
Acknowledgements--The authors wish to thank Dr C. A. Boulet and Mr A. S. Hansen for providing the organophosphate nerve agents used in these studies.
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