Morphological Effects of Neuropathy-Inducing Organophosphorus Compounds in Primary Dorsal Root Ganglia Cell Cultures

NeuroToxicology 24 (2003) 787–796

Morphological Effects of Neuropathy-Inducing Organophosphorus Compounds in Primary Dorsal Root Ganglia Cell Cultures Christiane Massicotte, Bernard S. Jortner, Marion Ehrich* Laboratory for Neurotoxicity Studies, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, 1 Duckpond Drive, Blacksburg, VA 24061-0442, USA Received 21 June 2002; accepted 25 March 2003

Abstract Chick embryo dorsal root ganglia (DRG) cultures were used to explore early pathological events associated with exposure to neuropathy-inducing organophosphorus (OP) compounds. This approach used an in vitro neuronal system from the species that provides the animal model for OP-induced delayed neuropathy (OPIDN). DRG were obtained from 9-day-old chick embryos, and grown for 14 days in minimal essential medium (MEM) supplemented with bovine and human placental sera and growth factors. Cultures were then exposed to 1 mM of the OP compounds phenyl saligenin phosphate (PSP) or mipafox, which readily elicit OPIDN in hens, paraoxon, which does not cause OPIDN, or the DMSO vehicle. The medium containing these toxicants was removed after 12 h, and cultures maintained for 4–7 days postexposure. Morphometric analysis of neurites was performed by inverted microscopy, which demonstrated that neurites of cells treated with mipafox or PSP but not with paraoxon had decreased length-to-diameter ratios at day 4 post-exposure. Ultrastructural alterations of neurons treated with PSP and mipafox included dissolution of microtubules and neurofilaments and degrading mitochondria. Paraoxon-treated and DMSO control neuronal cell cultures did not show such evident ultrastructural changes. This study demonstrates that chick DRG show pathological changes following exposure to neuropathy-inducing OP compounds. # 2003 Elsevier Science Inc. All rights reserved.

Keywords: Organophosphorus compounds; Dorsal root ganglia; Morphology; Morphometry

INTRODUCTION Some organophosphorus (OP) compounds can induce a delayed neuropathy (OPIDN), with resulting axonopathy progressing to myelinated fiber degeneration within peripheral nerves and spinal cord tracts of susceptible animals beginning 4–14 days after exposure. The pathology of OPIDN has been described in different animal species (rats, cats, monkey, sheep, and chickens) with toxicants such as tri-ortho-tolyl phosphate (TOTP), diisopropylfluorophosphate (DFP), phenyl saligenin phosphate (PSP) and mipafox, which appear to elicit similar lesions (Abou-Donia, 1981; * Corresponding author. Tel.: þ1-540-231-4621; fax: þ1-540-231-6033. E-mail address: [email protected] (M. Ehrich).

Bischoff, 1967; Bouldin and Cavanagh, 1979; Ehrich and Jortner, 2001; Prineas, 1969). Chickens are the most commonly used experimental animal for studies of OPIDN because, along with well-defined locomotor deficits, they exhibit distinct bilateral lesions in the spinal cord (mainly long tracts), brain stem, cerebellum, and peripheral nerves (Ehrich and Jortner, 2001; US Environmental Protection Agency (EPA), 1991). These lesions primarily involve larger myelinated axons, and include swelling, intra-axonal debris, loss of microtubules and neurofilaments (10 mm intermediate filaments), degenerated mitochondria and accumulation of vacuoles, progressing to axonal degeneration. Associated thinning or breakdown of myelin (with axonal swelling), along with astrocytic, Schwann cell and macrophage reactions, occurs in OPIDN. These lesions occur in chicks as well as in adult hens,

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although chicks are less susceptible to OPIDN (Funk et al., 1994). In addition to the morphological changes described in animal models with OPIDN, OP-induced cytotoxicity has been demonstrated in vitro (Carlson and Ehrich, 1999; Carlson et al., 2000; Mochida et al., 1988). In vitro systems have been used to examine the cellular mechanisms associated with the pathogenesis of OPIDN, as such studies are difficult in animal models. Neurotoxic OP compounds have elicited changes indicative of apoptosis, including fragmentation of DNA (Carlson and Ehrich, 1999; Carlson et al., 2000). OP compounds can inhibit metabolic processes and energy production, including glucose metabolism and adenosine incorporation into ATP (Harvey and Sharma, 1980; Knoth-Anderson et al., 1992). In addition, they can cause morphological changes associated with cell death within in vitro systems (Tuler and Bowen, 1989). Primary neuronal cultures have been used in neurotoxicology to assess effects of toxicants and nerve growth factors (NGFs). Evaluations included determination of length and diameter of neurites (Argiro et al., 1985; Kawa et al., 1998; Oorschot et al., 1991; Tosney and Landmesser, 1985; Ventimiglia et al., 1995; Yamada et al., 1971). These studies demonstrated the utility of such in vitro systems when measuring conformational neuronal changes. Although several morphometric investigations evaluated neuronal responses to toxicants in primary cell preparations, morphometric analysis has as yet not been performed for OP delayed neurotoxicants in this type of model system. The morphological effects of OP compound exposure on primary neuronal cell cultures from chicken embryos are reported here. The present study describes lesions in neuronal cell cultures from chicken dorsal root ganglia (DRG) after exposure to neuropathy-inducing OP compounds, using inverted and electron microscopic methods of imaging, and morphometric analysis of neurites. Although structural alterations in mitochondria have been previously demonstrated following OP exposure in other cell culture systems (Knoth-Anderson et al., 1992; Antunes-Madeira et al., 1994; Carlson and Ehrich, 1999), specific data related to structural effects following exposure to neuropathy-induced OP compounds in primary DRG cultures are unreported. The present study compared morphological changes in DRG cultures to those observed in vivo (Dyer et al., 1991, 1992; Ehrich and Jortner, 2001; Jortner and Ehrich, 1987; Jortner et al., 1989; Massicotte et al., 1999).

MATERIALS AND METHODS Organophosphorus Compounds Phenyl saligenin phosphate and mipafox were synthesized by Lark Enterprises (Webster, MA). These OP compounds were used because they induce delayed neuropathy in vivo (Ehrich and Jortner, 2001). Paraoxon (Chem Services, West Chester, PA) was used as a non-neuropathic OP control. Stock solutions containing PSP, mipafox, and paraoxon at 100 mM were prepared by dissolving these OP compounds in 90% dimethyl sulfoxide (DMSO). In this study, cytotoxic effects of the OP compounds were determined with a primary neuronal cell culture exposed to 1 mM concentrations of PSP, mipafox and paraoxon. Negative controls received DMSO (0.1%) vehicle only. The exposure time was 12 h, based on previous studies that demonstrated OP-induced effects on mitochondria in a neuroblastoma cell line in that period of time (Carlson and Ehrich, 1999). Neuronal Preparation from Dorsal Root Ganglia Cultures Dorsal root ganglia of 9-day-old chick embryos were dissected under sterile conditions from the lumbar intumescence (Blood, 1975; Kleitman et al., 1995). Under a dissecting microscope, the embryo was laid on its back and the abdominal viscera were removed with care to prevent damage of the DRG underneath. The lumbar DRGs were collected with microdissecting forceps, and transferred to a solution containing L15 medium (Sigma Cell Culture Products, St. Louis, MO). Strict sterile procedures were observed during dissections and in all subsequent steps of the purifying processes. Following DRG collection from approximately 30 embryos, the ganglia were incubated with 0.25% trypsin for 45 min at 37 8C on a rotatory shaker. Trypsin inactivation was achieved by the addition of L-15 medium supplemented with 15–20% fetal bovine serum (FBS). Then the sample was centrifuged at 80g for 5 min, and the pellets resuspended and triturated in 1 ml of serum-containing medium. The volume of this suspension was increased to 5 ml, recentrifuged as before, and resuspended in 10 ml of a solution containing Eagle’s minimum essential medium (E-MEM, Sigma Cell Culture Products) supplemented with 5% human placental serum (HPS, Scantibodies Laboratory Inc., Santee, CA) to decrease neuronal degeneration (Kleitman et al., 1995), 50 ng/ml

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of nerve growth factor (fraction 2.5S, murine, natural, Gibco BRL, Grand Island, NY), 5 mg/l g-irradiated and lyophilized bovine insulin (Sigma), 10 mg/l human transferrin (98% purity, Sigma), 20 nM progesterone (Sigma), 30 nM sodium selenite anhydrous (Johnson Matthew Electronics, Ward Hill, MA), and 100 mM tetramethylenediamine dihydrochloride (98% purity, Sigma). For inverted microscopic evaluation, 3 ml of this solution were placed in 35 mm round collagen coated plastic dishes. For electron microscopy, the cell suspension was placed in poly-D-lysine and collagen coated permanox plates. After the cells had been allowed to attach to the dishes for 12 h, the purification procedure was initiated by refeeding the cell culture with a solution containing antimitotic agents (E-MEM, 5% HPS, 498 mg/dl glucose, 50 ng/ml NGF, 10 mg/l transferrin, 100 mM putrescine, 20 nM progesterone, 5 mg/l bovine insulin, 30 nM sodium selenite, 10 mM uridine, 10 mM FdU). The antimitotic protocol was accomplished by alternating antimitotic feedings with maintenance feedings according to the following daily schedule: maintenance feeding on days 1, 4–6, 8–10 and 12 onward, and antimitotic feeding on days 2–4, and 6–8. Prior to performing experiments, the purified neuronal cultures were allowed to rest in maintenance medium for 1 week to ensure that no residual FdU remained. For morphological studies, the neuronal cells were exposed for 12 h to either 1 mM PSP, mipafox, or paraoxon. Medium containing OP compounds were removed at 12 h, and the culture was kept in the previously described maintenance medium for an additional 4– 7 days prior to analysis. Morphological Evaluation and Ultrastructural Examination Morphometric data were obtained using inverted microscopic images. Two culture dishes of living cells were used for each treatment (n ¼ 2), and several images were recorded from each treated dish (subsample, n ¼ 50), with the Oncor Image software system (Gaithersburg, MD). Following further magnification with Photoshop Software, neurites were outlined on the computer (n ¼ 50 per dish) by a microscopist blinded to the treatment status of the dish, and analyzed with Adobe Photoshop software (Morphometric Analysis Tool Kit, 3.0). Computerized parameters used for the evaluation of neurite integrity 4 and 7 days following OP exposures included the radial length, minimum, maximum and mean diameters, skew, kurtosis, and total area

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occupied by the neurites on the culture plate (Argiro et al., 1985; Kawa et al., 1998; Oorschot et al., 1991; Ventimiglia et al., 1995). The radial length was calculated from a line drawn parallel to the neurite. The mean neurite diameter was determined by taking the mean of 20 equally spaced diameter readings within the neurite of each neuron, measured at 908 to the neurite membrane. The maximum diameter represented the greatest neurite diameter recorded during calculations of the mean neurite diameter. Based on these values, length-to-diameter ratios representing diffuse neurite swelling (LD ¼ total neurite radial length/mean neurite diameter) and neurite segmental swelling (s ¼ [(maximum neurite diameter  mean neurite diameter)/mean neurite diameter]  100) were calculated for each neurite evaluated. Random sampling was used for estimation of all parameters (Mayhew, 1990). The cell area chosen was selected by dividing the 35 mm plastic round bottom dish into four equally partitioned quadrants. Cells from only one of the quadrants, selected prior to analysis, were evaluated for morphological studies (light and electron microscopy). The MIXED procedure of the SAS System (ver. 8.2, SAS Institute Inc., Cary, NC) was used to perform a mixed model repeated measures analysis of variance to test for effects of OP treatment, days in culture, and their interaction. The culture plates were the experimental unit with individual neurites as subsamples within a culture plate. For transmission electron microscopy, cultures fixed in the plates with 2% glutaraldehyde/1% paraformaldehyde in 0.07 M phosphate buffer, pH 7.4, were postfixed for 2 h in 1% OsO4 in 0.07 M phosphate buffer at pH 7.4, and washed with 0.1 M phosphate buffered solution. Following post-fixation, the tissues were dehydrated in graded concentrations of ethanol (30, 50, 70, 95, 100%), and cleared in propylene oxide and embedded by adding 3 ml epoxy resin to the plate. The plate was kept at 60 8C for 3 h before BEEM1 capsules (size 3) were placed over selected areas of cell growth. The plate was then polymerized at 60 8C for 24 h before the BEEM1 capsules were removed from the plates. The cells on the surface of the capsule were sectioned at 80–90 nm, and then placed on 200 mesh grids and stained with 2% uranyl acetate and lead citrate. These thin sections were examined with a JEOL JEM-100CX II transmission electron microscope. For each treated cell culture dish (n ¼ 2), 100–125 electron micrographs (neurites and perikaryon) were taken and evaluated by two observers, one of whom was blinded to the treatment groups. The morphological changes in

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neurons from the treated dishes, based on an agreement from both evaluators, were compared to controls. The electron microscopic findings were then compared to OP-induced axonal pathology observed in sections of nerves from chickens treated with neuropathy-inducing OP compounds (Dyer et al., 1991; Ehrich and Jortner, 2001; Jortner and Ehrich, 1987; Jortner et al., 1989; Massicotte et al., 1999).

RESULTS Morphometric Analysis of DRG Neurites Exposed to OP Compounds The predominant cytological element examined was a neuron made up of a cell body and a neurite (Fig. 1a). These neurites were single, long, thin processes resembling axons. Swelling was evident in portions of the neurites of cultures treated with mipafox and PSP (Fig. 1b). Morphometric analysis did not reveal statistically significant changes in mean diameter of neurites with time in culture (0:23  0:01 mm at day 4, and 0:24  0:07 mm at day 7 post-exposure to vehicle). While not affecting mean diameters, time in culture resulted in an increase in neurite length of OP-treated cultures (Fig. 2a). Treatment with neuropathy-inducing OP compounds (mipafox and PSP) decreased LD ratios determined 4 days after exposure (Fig. 2b). LD ratios for cultures treated with mipafox (LD ¼ 9:6  0:2) and PSP (LD ¼ 9:4  0:9) were lower than LD ratios of control neuronal populations (LD ¼ 14:9  2:6) and cells exposed to paraoxon (LD ¼ 12:5  0:4). This effect on LD was, however, transient, and not noted at post-exposure day 7. A tendency toward increased swelling of the neurites (s) was noted at day 7 postexposure, with s ¼ 42  5 in control cultures and s ¼ 61  2 and 51  4 in cells treated with paraoxon and mipafox, respectively (Fig. 2c). The frequency of axonal swelling in treated neurites was compared to controls using neurite area per unit length at 4 days after treatment. These values were significantly greater (P < 0:01) in neurites examined morphometrically following treatment with mipafox and PSP than in neurites of cells treated with paraoxon or DMSO. These ratios were 0.35 (n ¼ 53), 0.33 (n ¼ 72), 0.23 (n ¼ 70) and 0.23 (n ¼ 47), respectively. The percentages of neurites with values >1.7 times the standard deviation from the mean of neurites in control plates was 32% if exposed to mipafox, 26% if exposed to PSP, 6% if exposed to paraoxon, and 13% if exposed to DMSO.

Fig. 1. Light micrographs of chick DRG cultures representing those used for morphometric analysis. (a) Control at day 4 postdosing (0.1% DMSO) demonstrating neuronal cell bodies and their neurites. (b) Culture 4 days after a 12 h exposure to 1 mM mipafox showing prominent, diffuse swelling of the neurites compared to controls.

Electron Microscopic Evaluation Control and paraoxon-treated cultures revealed similar morphological features. The perikaryal cytoplasm was rich in organelles, with prominent polyribosomes, rough endoplasmic reticulum, vesicles, neurofilaments and mitochondria (Fig. 3a). Rough endoplasmic reticulum and most mitochondria appeared to end at the

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initial segment of the neurite. There were prominent collections of polyribosomes and ribosomes beneath the plasma membrane. The proximal neurite was also rich in neurofilaments. Neurite processes contained dense cores and pale vesicles, microtubules, and smooth endoplasmic reticulum. As the neurite extended distally from the cell body, decreased numbers of organelles (mitochondria, vesicles with dense and pale vesicles, smooth endoplasmic reticulum), fewer neurofilaments, and occasional swellings were observed (Fig. 3b). The swellings in small neuritis usually contained mitochondria. Definite presynaptic endings were not seen. Normal mitochondrial structures were present in these cultured neurons, including a smooth and regular double layer membrane surrounding lamellar, tubular and vesicular cristae within the more electron-dense matrix. Swellings of the distal regions of neurites were occasionally present, with increased filaments and dense core and clear vesicles (Fig. 3c). Cultures treated with the neuropathy-inducing OP compounds mipafox and PSP showed different morphological features than control cells or cells treated with paraoxon. By day 4 post-exposure, mipafox and PSP-treated cells had a reduction in microtubule-rich, thin neurites, so common in controls. In the cultures treated with these neuropathy-inducing OP compounds, the neurites often appeared thicker, with altered mitochondria. In addition, groups of neurites showed a sequence of increased separation and dissolution of microtubules and neurofilaments, and mitochondrial degeneration, progressing toward marked neurite swelling and degradation. Extensive cytologic debris was often detected in the PSP-treated cultures both 4 and 7 days after the 12 h dosing period (Fig. 4a). Unlike control cells, the neurite regions of PSP and

Fig. 2. Results of morphometric studies showing OP compound effects on DRG cultures 4 and 7 days post-exposure. (a) Effects of OP compounds on neurite radial lengths at days 4 and 7 days postexposure. Radial lengths were calculated for each neurite evaluated (n ¼ 2 plates per treatment, n ¼ 50 observations per plate). Radial lengths of the neurites were longer on day 7 than on day 4. Total radial lengths of neurites in the neuronal cultures

exposed to OP compounds were not significantly different than controls at days 4 and day 7 post-exposure. (b) Effects of OP compounds on the calculated LD ratios at days 4 and day 7 postexposure. Neurite radial length to diameter ratios (LD ¼ total radial length/mean diameter, LD ¼ mean  S:E:M:) were calculated from n ¼ 2 plates, n ¼ 50 observations per plate. The LD ratios were lower for cultures exposed to mipafox (LD ¼ 9:6  0:2) and PSP (LD ¼ 9:4  0:9) than control neuronal populations (LD ¼ 14:9  2:6) or cells treated with paraoxon (LD ¼ 12:5  0:4) at day 4 post-dosing. LD ratios of OP-treated cultures were significantly greater at day 7 post-dosing compared to day 4, and no significant differences were present among treatment groups at that time. (c) Effects of OP compounds were also determined as segmental swelling of the neurites (s ¼ [(maximum diameter  mean diameter)/mean diameter]  100) at days 4 and 7 post-exposure. Segmental swelling of neurites was noted 7 days post-exposure to paraoxon.

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Fig. 3. Control neuronal cells from DRG cultures. (a) The perikaryal cytoplasm is rich in organelles, with prominent polyribosomes, rough endoplasmic reticulum, vesicles, and mitochondria; 15,000. (b). A cluster of neurites which contain dense core and pale vesicles, mitochondria, and smooth endoplasmic reticulum. Regional swelling of neurites is present (arrow); 13,680. (c) Local swelling of a neurite, with dense core and clear vesicles, microtubules and filaments; 13,176.

mipafox-treated cells had numerous swellings along their course and at their terminals (Fig. 4a and b). Some regional neurofilamentous aggregates were seen. These changes progressed to lytic regions, neurite rupture and debris formation. Masses of membranous cytoplasmic bodies, or of microtubules and neurofilaments were noted in some degenerative swellings (Fig. 4b). Similar observations were made for mipafox-treated neurons, although changes were somewhat less severe. Neurons treated with mipafox or PSP showed more frequent and

more severe mitochondrial damage within their cell body and neurites than in the control populations (Fig. 4c and d). In the early stage of mitochondrial degeneration, swelling and progressive dissolution of the matrix were observed, with increased mitochondrial size. More severe signs of mitochondrial degeneration were frequently encountered, including disordered cristae, membranous debris, dense matrix containing some intra-matrical granules, and irregular membranes with blebbing (Fig. 4c and d).

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Fig. 4. Neuronal DRG cultures 4 days after a 12 h exposure to PSP or mipafox. (a) 1 mM PSP. Extensive cell debris is present (arrow). Viable neurites with numerous blebs (swellings) are seen (arrowheads); 9048. (b) 1 mM PSP. Swollen neurite containing masses of membranous cytoplasmic bodies; 20,801. (c) 1 mM PSP. There are regions of swelling in the neurites, with cytoskeletal lysis containing an altered mitochondrion (arrow); 45,820. (d) 1 mM mipafox. Mitochondrion with electron-dense matrix (arrow); 76,125.

DISCUSSION In this study organophosphate exposure caused visible evidence of cell swelling, decreases in length-to-diameter ratios of cultured chick dorsal root

ganglia neurites, and ultrastructural evidence of damage. The effects were more notable with neuropathy-inducing mipafox and PSP than following exposure to paraoxon, which does not cause OPIDN, suggesting that this culture system has promise for

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investigation of cellular and subcellular effects of OPIDN. Axonal degeneration of peripheral nerves is a prominent feature of OPIDN in chickens, a pathological state we have assessed in long axons of cultured primary peripheral neurons. Such cultures of chick dorsal root ganglia have been shown to achieve neuronal maturation, making them useful for neurobiological studies (Kleitman et al., 1995; Oorschot et al., 1991). They had not previously been used to investigate quantitative and qualitative morphological changes, including changes at the ultrastructural level that occur following exposure to neuropathy-inducing organophosphorus esters. Because the adult hen is considered the most reliable animal model for the study of OPIDN (Abou-Donia, 1981; Jortner and Ehrich, 1987; US Environmental Protection Agency (EPA), 1991), this in vitro neuronal cell model is valuable because it allows investigation of OP compounds in a target species. DRG were removed from chicken embryos and permitted to grow and develop relatively mature neurites in neuronal cultures. This primary cell culture preparation is the closest in vitro representation of neurons in vivo in a susceptible species, and was useful for studies of pathogenic mechanisms of OPIDN at the cellular level. In these studies we noted lower LD ratios at 4 days following a 12 h exposure to mipafox and PSP, when cultures were viewed by inverted light microscopy, than those of controls and paraoxon-treated cultures. Our dosing paradigm approximates the in vitro inactivation of OP compounds by biotransformation and the appearance of the initial lesions of OPIDN in peripheral nerves (El-Fawal et al., 1990). In contrast to previously published papers (Kawa et al., 1998; Ventimiglia et al., 1995), which included cell counts, neurite numbers and neurite perimeters, the space occupied by DRG cultures could not be divided into small regions as most neurite lengths in the present study exceeded previously recommended sampling field areas on the culture plate. (They were longer than a single field of view.) Therefore, the systematic sampling methods used to collect morphometric data in this study were modified. Because axonal shape is affected by Wallerian degeneration and distal swellings that are the predominant features in OPIDN, lengths, areas and diameters of neurites were chosen for evaluation using a computerized system. Skew and kurtosis were measured but did not provide additional useful information to differentiate among treatment groups. Time in culture and the presence of growth factors in the medium have been associated with increased neurite length and

likely affected the lack of observable differences in LD ratios at 7 days (Argiro et al., 1985; Deckworth, 1998; Kaplan and Miller, 2000; Ventimiglia et al., 1995) of OP-treated cultures. The lack of OP-induced inhibition of early growth of control cells may have contributed to the lesser difference in neurite growth noted between days 4 and 7 in those cultures. Electron microscopy performed on the DRG cultured neurons used in this study provided high resolution of the ultrastructural features of the perikaryon and neurite. This allowed assessment of the damage to neurites induced by neurotoxic OP compounds. Lesions of axons and their growth cones previously described in the literature were used as references for the present study (Bunge and Bunge, 1984; Cheville, 1983; Dyck et al., 1993; Ghadially, 1988; Gray, 1975; Peters et al., 1991). The results of this study suggest that the DRG cell model is more appropriate to study the effects of neuropathy-inducing compounds than previous models using immortalized cell lines, because the latter do not allow sufficient neurite outgrowth to provide an in vivo model of the axonal degeneration (Henschler et al., 1992; Nostrandt et al., 1992). Segmental neurite swellings were frequently observed in the neurons described in this study, which resembled the distal, non-terminal axonal degeneration that is a feature of OPIDN in animals (Bouldin and Cavanagh, 1979). Such neurite swellings in the present study were often associated with progressive dissolution of microtubules and neurofilaments, corresponding to the axonal cytoskeletal degeneration described as an important event in OPIDN in vivo (Bischoff, 1967; Prineas, 1969). The accumulation of membranous structures along the neurites in cultured DRG treated with neuropathy-inducing OP compounds is also a feature of the lesions of OPIDN in animal models (Bischoff, 1967; Bouldin and Cavanagh, 1979; Prineas, 1969). In addition, the current study provides evidence for a toxic effect of OP compounds on mitochondria as suggested previously (Carlson and Ehrich, 1999; Massicotte et al., 2001). In conclusion, the lesions observed in chick embryo DRG cell cultures exposed to neuropathy-inducing OP compounds had similarities to some of the axonal morphological changes described in hens with OPIDN. Since these cultures are derived from embryonic cells, future studies might use higher dosages or more prolonged exposures, because chicks are not as susceptible as adults to the effects of these toxicants (Funk et al., 1994). However, DRG cultures provide the most appropriate in vitro model for evaluation of the effects of neuropathy-inducing compounds at the cellular level.

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ACKNOWLEDGEMENTS The authors gratefully acknowledge the technical assistance of Virginia Viers, Kathy Lowe, Sandy Hancock, Kristel Fuhrman, Wen Li, and Daniel Ward without whom this work could not have been performed. Funds from Virginia-Maryland Regional College of Veterinary Medicine and its Laboratory for Neurotoxicity Studies supported this work. REFERENCES Abou-Donia MB. Organophosphorus ester-induced delayed neurotoxicity. Ann Rev Pharmacol Toxicol 1981;21:511–48. Antunes-Madeira MC, Videira RA, Madeira VMC. Effects of parathion on membrane organization and its implications for the mechanisms of toxicity. Biochim Biophys Acta 1994;1190: 149–54. Argiro V, Bunge MB, Johnson MI. A quantitative study of growth cone filopodial extension. J Neurosci Res 1985;13:149–62. Bischoff A. The ultrastructure of tri-ortho-cresyl phosphate poisoning. Part I. Studies on myelin and axonal alterations in the sciatic nerve. Acta Neuropath (Berl) 1967;9:158–74. Blood LA. Scanning electron microscope observations of the outgrowth from embryonic chick dorsal root ganglia in culture. Neurobiology 1975;5:75–83. Bouldin WT, Cavanagh JB. Organophosphorous neuropathy. Part II. A fine-structural study of the early stages of axonal degeneration. Am J Pathol 1979;94:253–61. Bunge RP, Bunge MB. Tissue culture observations relating to peripheral nerve development, regeneration, and disease. In: Dyck PJ, Thomas PK, Lambert EH, Bunge RP, editors. Peripheral neuropathy. Philadelphia: Saunders; 1984. p. 378–99. Carlson K, Ehrich M. Organophosphorus compound-induced modification of SH-SY5Y human neuroblastoma mitochondrial transmembrane potential. Toxicol Appl Pharmacol 1999;160: 33–42. Carlson K, Jortner BS, Ehrich M. Organophosphorus compoundinduced apoptosis in SH-SY5Y human neuroblastoma cells. Toxicol Appl Pharmacol 2000;168:102–13. Cheville NF. Cell degeneration. In: Cheville NF, editor. Cell pathology. 2nd ed. Ames, IA: The Iowa State University Press; 1983. p. 114–29. Deckworth TL. Molecular mechanisms of neuroprotection from neuronal death by trophic deprivation. In: Mattson MR, editor. Neuroprotective signal transduction. Totawa, NJ: Humana Press; 1998. p. 61–82. Dyck PJ, Giannini C, Lais A. Pathologic alterations of nerves. In: Dyck PJ, Thomas PK, editors. Peripheral neuropathy. Philadelphia, PA: Saunders; 1993. p. 514–97. Dyer KR, El-Fawal HAN, Ehrich MF. Comparison of organophosphate-induced delayed neuropathy between branches of the tibial nerve and the biventer cervicis nerve in chickens. Neurotoxicology 1991;12:687–96. Ehrich M, Jortner BJ. Organophosphorus-induced delayed neuropathy. In: Kreiger R, editor. Handbook of pesticide toxicology, vol. 2, Agents. San Diego: Academic Press; 2001. p. 987–1012.

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El-Fawal HAN, Jortner BS, Ehrich M. Use of the biventer cervicis nerve-muscle preparation to detect early changes following exposure to organophosphates inducing delayed neuropathy. Fundam Appl Toxicol 1990;15:108–20. Funk KA, Henderson JD, Liu CH, Higgins RJ, Wilson BW. Neuropathology of organophosphate-induced delayed neuropathy (OPIDN) in young chicks. Arch Toxicol 1994;68: 308–16. Ghadially FN. Mitochondria, microtubules. In: Ghadially FN, editor. Ultrastructural pathology of the cell and matrix. 3rd ed. London: Butterworths; 1988. p. 191–328, 937–50. Gray EG. Presynaptic microtubules and their association with synaptic vesicles. Roy Soc B 1975;190:369–72. Harvey MJ, Sharma RP. Organophosphate cytotoxicity: the effects on protein metabolism in cultured neuroblastoma cells. J Environ Pathol Toxicol 1980;3:423–36. Henschler D, Schmuck G, van Aerssen M, Schiffmann D. The inhibitory effect of neuropathic organophosphate esters on neurite outgrowth in cell cultures: a basis for screening for delayed neuropathy. In Vitro Toxicol 1992;6:327–35. Jortner BS, Ehrich M. Neuropathological effects of phenylsaligenin phosphate in chickens. Neurotoxicology 1987;8: 97–108. Jortner BS, Shell L, El-Fawal H, Ehrich M. Myelinated nerve regeneration following organophosphorus ester-induced delayed neuropathy. Neurotoxicology 1989;10:717–26. Kaplan DR, Miller FD. Neurotrophin signal transduction in the nervous system. Curr Opin Neurobiol 2000;10:381–91. Kawa A, Stahlhut M, Berezin A, Bock E, Berezin V. A simple procedure for morphometric analysis of processes and growth cones of neurons in culture using parameters derived from the contour and convex hull of the object. J Neurosci Methods 1998;79:53–64. Kleitman N, Wood PM, Bunge RP. Tissue culture methods for the study of myelination. In: Banker G, Goselin K, editors. Culturing nerve cells. Cambridge: Massachussetts Institute of Technology; 1995. p. 545–94. Knoth-Anderson J, Veronesi B, Jones K, Lapadula DM, AbouDonia MB. Triphenyl phosphite-induced ultrastructural changes in bovine adrenomedullary chromaffin cells. Toxicol Appl Pharmacol 1992;112:110–9. Massicotte C, Inzana KD, Ehrich M, Jortner BS. Neuropathologic effects of phenylmethylsulfonyl fluoride (PMSF)-induced promotion and protection in organophosphorus ester-induced delayed neuropathy (OPIDN) in hens. Neurotoxicology 1999; 20:749–60. Massicotte C, Barber DS, Jortner BS, Ehrich M. Nerve conduction and ATP concentrations in sciatic-tibial and medial plantar nerves of hens given phenylsaligenin phosphate. Neurotoxicology 2001;22:91–8. Mayhew TM. Efficient and unbiased sampling of nerve fibers for estimating fiber number and size. In: Conn PM, editor. Quantitative and qualitative microscopy. San Diego, CA: Academic Press; 1990. p. 172–87. Mochida K, Gomyoda M, Fujita T, Yamagata K. Tricresyl phosphate and triphenyl phosphate are toxic to cultured human, monkey and dog cells. Zbl Batk Hyg B 1988;185: 427–9. Nostrandt AC, Rowles TK, Ehrich M. Cytotoxic effects of organophosphorus esters and other neurotoxic chemicals on cultured cells. In Vitro Toxicol 1992;5:127–36.

796

C. Massicotte et al. / NeuroToxicology 24 (2003) 787–796

Oorschot DE, Peterson DA, Jones DG. Neurite growth from, and neuronal survival within cultured explants of the nervous system: a critical review of morphometric and stereological methods, and suggestions for the future. Prog Neurobiol 1991; 37:525–46. Peters A, Palay SL, Webster HF. The axon. In: Peters A, editor. The fine structure of the nervous system. New York: Oxford University Press; 1991. p. 101–37. Prineas J. The pathogenesis of dying-back polyneuropathies. Part I. An ultrastructural study of experimental tri-ortho-cresyl phosphate intoxication in the cat. J Neuropathol Exp Neurol 1969;28:571–97. Tosney KW, Landmesser LT. Growth cone morphology and trajectory in the lumbosacral region of the chick embryo. J Neurosci 1985;5:2345–58.

Tuler SM, Bowen JM. Toxic effect of organophosphates on nerve cell growth and ultrastructure in culture. J Toxicol Environ Health 1989;27:209–23. US Environmental Protection Agency (EPA). Pesticide assessment guidelines, subdivision F. Hazard evaluation: human and domestic animals. Addendum 10: Neurotoxicity, series 81, 82, 83. Springfield, VA: National Technical Information Service; 1991. p. 1–60. Ventimiglia R, Jones BE, Moller A. A quantitative method for morphometric analysis in neuronal cell culture: unbiased estimation of neuron area and number of branch points. J Neurosci Methods 1995;57:63–6. Yamada KM, Spooner BS, Wessells NK. Ultrastructure and function of growth cones and axons of cultured nerve cells. J Cell Biol 1971;49:614–35.