Reaggregate Cultures Research Articles

Validation ofIn VitroTests in Neurotoxicology

The major challenges in neurotoxicity testing relate to the complexity of the nervous system, the diversity of cell types involved, and the level of integration in the mammalian nervous system. In addition, compounds which have selective pharmacological effects as receptor agonists/antagonists (for example, strychnine) may be neurotoxic at non-cytotoxic concentrations. Tests should answer the following questions: a) when is an effect toxic?, b) when is a substance to be considered toxic? and c) is the long-term risk assessment valid?The two major strategies used in the development of in vitro neurotoxicity tests are: mechanistic, in which an attempt is made to elucidate the biochemical processes involved in neurotoxicity, and disease based, which could ultimately be the most useful strategy but which is currently constrained by lack of knowledge of the aetiology of most neurological illnesses. Potential in vitro test systems which are being developed include: a) simple tests which measure the activity of an enzyme (for example, acetylcholine esterase); b) studies involving single cell type culture (for example, neuroblastomas or dorsal root ganglion cells); c) complex primary co-culture systems (for example, reaggregate culture; 1, 2); and d) combinations of these, including tiered testing and battery testing (3). Unfortunately, the more complex a system is, the more extensive is the characterisation needed, and, arguably, such systems will never fully mimic the intact central nervous system/peripheral nervous system (CNS/PNS).To demonstrate some of the problems inherent in neurotoxicity test development, the use of a system which involves the inhibition of outgrowth is described. The ideas are developed to include the shift toward proliferation and/or apoptosis of non-terminally differentiated neurons.The most realistic objective for the optimised, integrated and validated in vitro reductionist approach for neurotoxicological assessment is for the screening of new compounds in parallel with: a) the in vivo holistic approach (for example, to obtain pharmacokinetics and absorption/receptor-binding data); and b) quantitative structure activity relationships (QSARs).This is necessarily a selective review, and more details of methodologies and strategies are presented in other publications (3, 4).

Glutamate release evoked by glutamate receptor agonists in cultured chick retina cells: Modulation by arachidonic acid

We studied the effect of ionotropic glutamate receptor agonists on the release of endogenous glutamate or of [3H]D-aspartate from reaggregate cultures (retinospheroids) or from monolayer cultures of chick retinal cells, respectively. Kainate increased the fluorescence ratio of the Na+ indicator SBFI and stimulated a dose-dependent release of glutamate in low (0.1 mM) Ca2+ medium, as measured using a fluorometric assay. Under the same experimental conditions, the release evoked by N-methyl-D-aspartate (NMDA; 400 microM) was about half of that evoked by the same kainate concentration; alpha-amino-3-hydroxy-5-methyl-4-isoxasolepropionic acid (AMPA; 400 microM) did not trigger a significant response. In the presence of 1 mM CaCl2, all of the agonists increased the [Ca2+]i, as determined with the fluorescence dye Indo-1, but the glutamate release evoked by NMDA and kainate was significantly lower than that measured in 0.1 mM CaCl2 medium. Inhibition by Ca2+ of the kainate-stimulated release of glutamate was partially reversed by the phospholipase A2 inhibitor oleiloxyethyl phosphorylcholine (OPC), suggesting that the effect was mediated by the release of arachidonic acid, which inhibits the glutamate carrier. Accordingly, kainate, NMDA, and AMPA stimulated a Ca(2+)-dependent release of [3H]arachidonic acid, and the direct addition of the exogenous fatty acid to the medium decreased the release of glutamate evoked by kainate in low (0.1 mM) CaCl2 medium. In monolayer cultures, we showed that NMDA, kainate, and AMPA also stimulated the release of [3H]D-aspartate, but in this case release in the presence of 1 mM CaCl2 was significantly higher than that evoked in media with no added Ca2+. The ranking order of efficacy for stimulation of Ca(2+)-dependent release of [3H]D-aspartate was NMDA > > kainate > AMPA.

Bone marrow-derived cells fail to induce positive selection in thymus reaggregation cultures.

The requirements for inducing positive selection of T cells were examined in thymus reaggregation cultures, a system in which dispersed populations of immature CD4+8+ cells and purified thymic epithelial cells (TEC) are reaggregated in tissue culture. Studies with TEC from mice selectively lacking major histocompatibility complex (MHC) class I (I-II+), class II (I+II-), or both class I and II (I-II-) molecules showed that class II expression was essential for the differentiation of CD4+8+ cells into CD4+8- cells. Unexpectedly, the generation of TCRhi CD4-8+ cells from CD4+8+ cells was apparent with I-II+ TEC but not with I-II- TEC, perhaps reflecting cross-reactive specificity of CD4-8+ cells for class II molecules. Significantly, the failure of I-II- TEC to generate TCRhi CD4+8- or CD4-8+ cells could not be overcome by adding MHC+ bone marrow-derived cells. These findings, together with experiments on purified subsets of TEC, suggest that positive selection in thymus reaggregation cultures is an exclusive property of cortical TEC.

A role for CD81 in early T cell development.

Early stages of T cell development are thought to include a series of coordinated interactions between thymocytes and other cells of the thymus. A monoclonal antibody specific for mouse CD81 was identified that blocked the appearance of alpha beta but not gamma delta T cells in fetal organ cultures initiated with day 14.5 thymus lobes. In reaggregation cultures with CD81-transfected fibroblasts, CD4-CD8- thymocytes differentiated into CD4+CD8+ T cells. Thus, interactions between immature thymocytes and stromal cells expressing CD81 are required and may be sufficient to induce early events associated with T cell development.

Thymic selection and cell division.

Cell division during thymic selection was studied with a system in which purified populations of T cell antigen receptor (TCR)- CD4+8+ (double-positive [DP]) cells and fetal thymic epithelial cells (TEC) were reaggregated in tissue culture. In this system, immature DP cells differentiate into mature single-positive (SP) CD4+8- and CD4-8+ TCRhi cells within 3-4 d, indicative of positive selection. By adding the DNA precursor, bromodeoxyuridine, to the cultures and staining cells for bromodeoxyuridine incorporation, T cell division in reaggregation cultures was found to be high on day 1, low on day 2, and high on days 4-5. Cell separation studies established that cell division on day 1 was restricted to DP blast cells. In the absence of blast cells, small DP cells failed to proliferate and differentiated into SP cells without cell division, thus indicating that proliferation is not an essential component of positive selection. This applied to SP cells generated within the first 2-3 d. Surprisingly, the SP cells generated later in culture showed a high rate of cell division; the proliferating SP cells were TCRhi and included both CD4+8- and CD4-8+ cells. Turnover of TCRhi SP cells was also prominent in the normal neonatal thymus and in TEC reaggregation cultures prepared with adult lymph node T cells. We speculate that division of mature SP cells in the perinatal thymic microenvironment is driven by stimulatory cytokines released from TEC. Such proliferation could be a device to expand the mature T cell repertoire before export to the periphery.

Glutamine synthetase activity as a marker of toxicity in cultures of embryonic chick brain and retina cells

With the goal of developing a fast and sensitive primary cell culture assay for the determination of neurotoxic potential of compounds, the effect of various toxins on the morphology, cell number (estimated as total cell protein), and glutamine synthetase activity of chick embryonic neural cells has been tested. Isolated retina or brain cells, grown as reaggregates in suspension cultures or as monolayers in 24-well plates, were treated with the substances from day 2 to day 6 after the start of culture. Concentrations causing 50% reduction in protein content in brain cell monolayers were as follows: MeHgCl (0.8 μ m), CdCl 2 (1 μ m), 3-acetyl pyridine (0.1 m m), penicillin (above 0.1 m m), diazepam (0.25 m m), acrylamide (0.3 m m), 2,4,5-T (0.8 m m), lindane (1 m m). In general, retina cells were more sensitive than brain cells. The reaggregate cultures were less sensitive to 1-methyl-4-phenylpyridinium ion (MPP +) and cadmium than monolayer cultures, which may be attributable to their metabolic stability or to diffusional limitations. Glutamine synthetase (GS) activity, measured as glutamate production from glutamine, was a more sensitive indicator of toxicity than total protein. Retinal cells grown as reaggregates or monolayer cultures, produced two to four times more glutamate than brain cells grown in a similar fashion. This indicates that retinal glial cell (Müller cell) differentiation proceeds in vitro faster than brain astrocyte differentiation, which is consistent with the in vivo developmental pattern. In some cases (methylmercury, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, MPP +, 3-acetyl pyridine, and lindane) a significant increase (as much as 30% of the basal level) was seen in the GS units/μg cell protein at concentrations of toxins below that reducing total cell protein. Thus, generation of neurotoxic glutamate might play a role in the cell destruction caused by the chemicals. Other substances (e.g. diazepam and cadmium) decreased the GS activity considerably, relative to decreases in total protein. This suggests that these xenobiotics act in a more general fashion to reduce metabolic activity and viability.

A serum‐free, pyruvate‐free medium that supports neonatal neural/glial cultures

Tissue culture media with serum generally cause excessive astrocyte proliferation in neonatal brain cultures, and often fail to support neonatal neurons. Published serum-free media for brain cultures contain sodium pyruvate, which interferes with lactate dehydrogenase (LDH) assays for cell death. We wanted to use neonatal neural-glial cultures in LDH assays while avoiding astrocyte proliferation, so we developed a serum-free medium without sodium pyruvate. Our initial medium was based on that of Romijn et al. (J Neurosci Methods 23:75-83, 1988), testing selected additives. Cell survival in 8-10-day-old cultures was measured using 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). N-acetylcysteine, citrate, superoxide dismutase, ascorbate, supplemental amino acids, and high levels of transferrin improved survival. The optimized medium supported neonatal brain cells in reaggregates or in monolayers of 400 cells/mm2 for several weeks with large, healthy-appearing neurons and very little astrocyte proliferation. Neurons stained strongly for the neuronal marker class III beta-tubulin and the synapse marker synaptophysin. Electron microscopy of reaggregate cultures demonstrated abundant neurons with synapses in a dense neuropil. This medium will be useful for various in vitro applications, especially those using LDH assays or requiring the use of neonatal cells.

The reeler gene-associated antigen on cajal-retzius neurons is a crucial molecule for laminar organization of cortical neurons

In the neurological mutant mouse reeler, the histological organization of the neocortex develops abnormally and essentially results in an inversion of the relative positions of the cortical layers. The reeler mutation, therefore, provides an insight into the molecular mechanisms underlying the formation of the cortical layers. We have generated a monoclonal antibody (CR-50) that probes a distinct allelic antigen present in wild-type but not in reeler mutant mice. CR-50 reacted specifically with Cajal-Retzius neurons, one of the first cortical neurons to differentiate in the neocortex, but whose functional role is not known. When dissociated cerebral cortical cells were incubated with CR-50 in reaggregation culture, the genotype-dependent histogenetic assembly of wild-type cortical cells resembled that of reeler mutants. These findings revealed that the selective expression of a distinct molecule on Cajal-Retzius neurons is critical for the normal lamination of cortical neurons in the mammalian neocortex.

Examination of developmental neurotoxicity by the use of tissue culture model systems.

1. The rotation-mediated three-dimensional reaggregate culture system is uniquely suited for studies on developmental neurotoxicity. In this system, it is possible to reconstruct central neuronal pathways and follow their development. 2. Exposure to drugs of abuse including methamphetamine and methylenedioxyamphetamine or the appetite suppressant, fenfluramine, reduces monoamines in the cultures in a dose-dependent manner and interrupts normal monoaminergic development. 3. While the monoaminergic neurones may attain normal rates of development following drug removal, the affected neurones are not capable of overcoming the drug-induced insults and a deficiency in monoamines persists throughout development. 4. In addition, the production of immortalized monoclonal hybrid cells obtained by fusion of fetal mesencephalic neurones with a neuroblastoma has yielded cell lines expressing a dopaminergic phenotype. 5. Such cells have been useful in establishing the relationship of neurotoxicity to cell lineage and can serve as models for the study of the cellular and molecular mechanisms of neurotoxicity.

Free-radical mediated toxicity in whole rat brain reaggregate cultures is prevented by alpha-tocopherol.

Free-radical mediated toxicity in whole rat brain reaggregate cultures is prevented by alpha-tocopherol.

Rapid stimulation of type I 5′-deiodinase in rat pituitaries by 3,3′,5-triiodo- l-thyronine

The negative feedback control of thyrotropin production by the anterior pituitary involves local 5′-deiodination of l-thyroxine (L-T 4) to the active thyroid hormone 3,3′,5-triiodo- l-thyronine (T 3) by two 5′-deiodinase isozymes which are distinctly regulated by thyroid hormone. T 3 rapidly increased steady-state mRNA levels and activity of type I iodothyronine-5′-deiodinase (5′DI) in rat anterior pituitary and in reaggregate cultures of anterior pituitaries. Type II 5′-deiodinase activity determined in parallel with 3,3′,5′-triiodo- l-thyronine (rT 3) as substrate was markedly lower than that of 5′DI. 5′DI mRNA levels and activity were higher in anterior pituitaries of female compared to male rats. Neither gender differences nor T 3 stimulation of 5′DI activity were found in the posterior part. These data demonstrate a T 3 dependent expression of 5′DI in euthyroid anterior pituitary and suggest that this isozyme serves a major function within the complex network of thyroid hormone homeostasis.

Positive Selection of Thymocytes

The requirement for immature thymocytes to demonstrate that their surface αβ T cell receptors (TCRs) have some reactivity to self-MHC class I or class I1 molecules is the barrier enforced by positive selection. Positively selected thymocytes are not driven to proliferate, and their receptors do not mutate away from self-recognition. Instead, cells are rescued from programmed cell death on the basis of self-recognition and go on to serve in the periphery as self-tolerant T cells. This different response in immature versus mature T cells to recognition of antigen through an unmutated TCR may be controlled by poorly understood changes in the trigger ability of the cells. Most αβ TCR thymocytes simply do not pass this test. Cells that initially fail positive selection may briefly continue to rearrange TCR a chain genes and finally succeed in expressing a selectable TCR. However, most DP thymocytes undergo a programmed cell death, ensuring that the selected repertoire is reactive with foreign peptides presented by self- MHC molecules. The nature of the signal triggered by the TCR-self-MHC interaction that rescues thymocytes from death remains an enigma. The interaction between the TCR and its MHC ligands that stimulates positive selection in fetal thymus organ culture (FTOC) is so weak that it does not stimulate any measurable signaling in mature T cells. For this reason, mature T cells cannot serve as model systems for the signal transduction pathway operating in thymocytes selection as obtained from the ever-expanding use of genetically manipulated animal model systems to elucidate the role of various gene products in thymocytes selection. The second source is the use of in vitro reaggregate culture systems that allow the study of positive selection from separated stromal and stem-cell components, in which it may soon be possible to introduce probes directly into the DP stem cells for positive selection.

Oxidative stress decreases antioxidant enzyme activities in reaggregation cultures of rat brain cells

The brain has been suggested to be especially sensitive to damage by reactive oxygen species. In this study, we examined the effects of hyperoxic conditions on the activities and mRNA levels of antioxidant enzymes in reaggregation cultures of rat forebrain cells. Cultures were exposed to 80% oxygen for 12–60 h starting on Days 17 and 33 in culture. Superoxide dismutase activities and mRNA levels were not affected by hyperoxia, whereas catalase activity was slightly decreased after 24 h in 80% oxygen at Day 17. Glutathione peroxidase activity was markedly decreased already after 12 h of hyperoxia, and decreased activities of glutathione reductase and glucose-6-phosphate dehydrogenase were also noted. The glutathione peroxidase mRNA levels were increased in hyperoxic cultures at Day 17 but not at Day 33. These results suggest that the enzymatic defense mechanisms against reactive oxygen species in the brain are rather weak and deteriorate during oxidative stress but that a potential for compensatory upregulation exists at least during the first postnatal weeks.

Study of the cardiotoxic potential of pharmaceutical compounds in chick myocardial myocyte reaggregate cultures

Cardiotoxicity produced by doxorubicin in vivo is considered to be due to a direct effect on the myocardium and this is also a major component with toxicity of isoprenaline and digoxin. In the case of the cardiotoxicity produced at high doses by the antihypertensives hydralazine and pinacidil, an indirect mechanism operating by way of their exaggerated pharmacological effects is believed to be responsible. These compounds were examined for their cardiotoxic potential in vitro using chick myocardial myocyte reaggregate (MMR) cultures; allylamine HCl was used as a positive control. Cultures were incubated for up to 24 hr with each compound; parameters analysed were: spontaneous beating activity (SBA), lactate dehydrogenase (LDH) leakage and microscopic evidence of cytotoxicity. Allylamine, doxorubicin, digoxin and to a lesser extent isoprenaline were highly toxic to MMR cultures, as demonstrated by their effects on SBA, LDH leakage and morphology. Hydralazine showed very mild cytotoxicity at the highest concentrations with no LDH leakage; pinacidil was not cytotoxic but showed a dose-related inhibition of SBA. These results confirm the direct toxic action of doxorubicin and digoxin on myocardial cells and indicate that this is also an important mechanism in vivo for isoprenaline. The lack of any significant toxicity with hydralazine and pinacidil accords with an indirect mechanism based on their pharmacology.

Ethanol‐Induced Increase in Catalase Activity in Reaggregation Cultures of Rat Brain Cells is Due to Increased Oligodendrocyte Differentiation

Recent studies have shown that ethanol exposure of reaggregation cultures of fetal rat brain cells causes an increased activity and amount of catalase, and also an increased activity of the oligodendrocyte marker enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP). In the present study, reaggregation cultures were grown in the presence of 40 mM ethanol during 17 days, corresponding to a period in vivo from gestational day 17 to postnatal day 12. The activities of catalase, the peroxisomal marker enzyme acyl-coenzyme A oxidase and CNP were increased in ethanol-treated cultures. Immunofluorescence experiments confirmed that catalase mainly resided in oligodendrocytes. By immunoperoxidase staining for CNP followed by image analysis, it was observed that the proportion of CNP-positive cells, as well as the intensity of staining, was increased in ethanol-treated cultures. Thus, the previously reported increase in catalase protein and activity in ethanol-treated cultures might be due to a stimulation, or earlier onset, of oligodendrocyte differentiation.

Differential expression of Mtv loci in MHC class II-positive thymic stromal cells.

Despite the important role played by endogenous superantigen in shaping the T cell repertoire, little is known concerning the expression of the different Mtv loci in cells of the thymic microenvironment involved in repertoire selection. Here we have examined the expression of a panel of Mtv Ags by different MHC class II+ stromal cel types using reverse transcriptase-PCR and monitored the effects of these stromal cells on the development of cells expressing Mtv-reactive TCR V beta elements in closed thymic organ culture systems. Although Mtv-6 and Mtv-8/9 mRNAs are expressed in normal thymus lobe organ cultures, no Mtv expression was detected in MHC class II+ thymic epithelial cells. In contrast a striking pattern of differential expression was observed in dendritic cells of thymic origin that were devoid of Mtv-8/9 but expressed readily detectable levels of Mtv-6. This pattern of Mtv gene expression correlated well with TCR V beta repertoire development. TCRV beta 3+ T cells, normally deleted in response to Mtv-6, were virtually absent from the single positive thymocyte compartment in thymic organ cultures where dendritic cells are present but were present in reaggregate cultures where the only MHC class II-positive cells were thymic epithelial cells. On the other hand, V beta 11+ T-cells were not deleted in organ cultures, possibly reflecting the absence of Mtv-8/9 expression in dendritic cells. Our studies suggest that the influence Mtvs have on shaping the T cell repertoire not only depends on their expression within a particular strain but also on their tissue specific expression in relation to MHC class II, which is necessary for their presentation.

The Neurotoxicity of Organic Solvents Studied Using Synaptosomes and Neural Cell Cultures

The most common acute neurotoxic effect of organic solvents is their central nervous system (CNS) depressant effect. The molecular mechanism underlying this effect is not known. The purpose of our studies has been to evaluate the adverse effects of organic solvents on the CNS in vitro. Synaptosomal membranes, whole brain reaggregate and astrocyte cultures were studied. Our results suggest that cell membrane integral proteins are targets for solvent impact, but that there are differences among various cell types. In addition to lipophilicity, the structure of solvent molecules seems to be important when considering CNS toxicity. Organic solvents increase the fluidity of the membranes, which may disturb the lipid-protein interaction and the optimal functioning of the enzyme. However, direct effects of solvents on proteins cannot be excluded. In vitro cell models can be used in methods designed to predict acute neurotoxic effects of foreign compounds, and in studies of neurotoxic mechanisms.

Daudi supernatant, unlike other H-Y antigen sources, exerts a sex-reversing effect on embryonic chick gonad differentiation

In vitro cultures of intact chick gonads (organ cultures) and reaggregation cultures of dispersed gonad cells (roller cultures) were made. Gonads or gonad cells from 7-day-old chick embryos, at the stage when sex-specific differentiation begins, were cultured in the presence of presumed H-Y antigen-containing supernatants, or co-cultured in the presence of H-Y antigen-producing cell lines. The H-Y antigen-producing cells tested were of human, mouse, bovine and chicken origin. During organ culture, addition of supernatant of the human lymphoma cell line Daudi, or co-culture with Daudi cells, stimulated a clear proliferation of the germinal epithelium in male gonads, indicating feminization. A similar effect was obtained by treatment with estradiol. In reaggregation culture, the increase in nuclear size of germ cells was chosen as a parameter for feminization. A significant increase of germ cell nuclear size was observed in gonads cultured in the presence of Daudi supernatant. In both organ cultures and reaggregation cultures, other tested H-Y antigen sources and semi-purified H-Y antigen fractions did not exert significant effects on differentiation of the gonads or on the average area of the germ cell nuclei. These findings suggest that it is not H-Y antigen, but another protein produced by Daudi cells, that might be responsible for the sex-reversing effects.

Avian Embryonic Brain Reaggregate Culture System .: I. Characterization for Organophosphorus Compound Toxicity Studies

An avian reaggregate culture system was characterized biochemically and morphologically for use in acute and chronic organophosphorus compound (OP) toxicity studies. Ten-day-old chick embryo brains were dissociated, reaggregated. and maintained in a chemically defined, serum- and antibiotic-free media. Acetylcholinesterase (ACHE), neuropathy target esterase (NTE), and 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP) were examined due to inhibition of these enzymes as a result of acute OP toxicity (ACHE) or delayed toxicity (NTE, CNP). The selected enzymes also indicate reaggregate neuronal (ACHE, possibly NTE), oligodendroglial (CNP), and astrocytic (glutamine synthetase (GS)) activities. Enzyme activities were compared to those in age-matched chick embryo and hatched chick brains. Reaggregate ACHE specific activity was similar to or higher than that of chick embryo or hatched chick. Reaggregate NTE specific activity was initially similar to that of 10-day-old chick embryo, and then increased but subsequently averaged 7.8 nmol/min/mg protein. In chick brain, NTE peaked at hatching and averaged 28 nmol/min/mg protein thereafter. Reaggregate CNP specific activity ranged from 103 to 426 nmol/min/mg protein, whereas activity gradually increased in chick embryo brain to an average of 140 nmol/min/mg protein posthatching. The mean GS activity ranged from 0.15 (Culture Day 4) to 1.09 nmol/min/mg protein (Culture Day 62). Mean protein values per flask ranged from 2.47 to 7.58 mg. Ultrastructurally, myelination was detected at Culture Day 7 and synapses at Day 6. The biochemical and ultrastructural features demonstrate that this reaggregate culture is a practical and sensitive in vitro system for studying both the acute and the long-term neurotoxicological effects of organophosphorus compounds.

Avian Embryonic Brain Reaggregate Culture System .: II. NTE Activity Discriminates between Effects of a Single Neuropathic or Nonneuropathic Organophosphorus Compound Exposure

Biochemical responses after a single exposure to either a neuropathic or a nonneuropathic organophosphorus compound (OP) were compared using chick embryonic brain cell reaggregates. Ten-day-old chick embryo brains were dissociated and then reaggregated and maintained in a chemically defined, serum-free medium without antibiotics. Seven days later, these cultures were treated for 20 min with either neuropathic diisopropyl phosphorofluoridate (DFP, 10 −4 M) or nonneuropathic paraoxon (10 −6 M). Reaggregates were assayed for acetylcholinesterase (ACHE), neuropathy target esterase (NTE), and 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP) activities for up to 32 days after exposure. These enzymes were examined due to inhibition of activity as a result of acute OP toxicity (ACHE) or delayed toxicity (NTE, CNP). DFP inhibited >95% of NTE activity immediately after exposure. By Postexposure Day 2, NTE specific activity was 22% of untreated activity but was similar to the untreated group levels by Postexposure Day 7. Paraoxon exposure did not affect NTE activity. Both paraoxon and DFP inhibited >99% of ACHE activity immediately after exposure. By Postexposure Day 2, ACHE specific activity in paraoxon-exposed cultures had recovered while ACHE remained 56% inhibited in DFP-exposed cultures. Both paraoxon- and DFP-exposed cultures recovered ACHE activity immediately following OP exposure if treated postexposure with an oxime reactivator, 2-pralidoxime. CNP specific activity was not affected by either paraoxon or DFP. These results demonstrated distinct differences in reaggregate NTE and ACHE activities after single exposure to neuropathic DFP and nonneuropathic paraoxon similar to those in avian in vivo assays.