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The effects of carbaryl and trichlorphon on differentiating mouse N2a neuroblastoma cells
Toxicology Letters 110 (1999) 79 – 84 www.elsevier.com/locate/toxlet
The effects of carbaryl and trichlorphon on differentiating mouse N2a neuroblastoma cells John Flaskos a, Maxine J. Fowler b, Cedric Teurtrie b, Alan J. Hargreaves b,* a
Laboratory of Biochemistry and Toxicology, Faculty of Veterinary Medicine, Aristotelian Uni6ersity of Thessaloniki, Thessaloniki, Greece b Department of Life Sciences, Nottingham Trent Uni6ersity, Clifton Lane, Nottingham NG11 8NS, UK Received 22 September 1998; received in revised form 25 July 1999; accepted 27 July 1999
Abstract The ability of the carbamate pesticide carbaryl (CB) and the organophosphate pesticide trichlorphon (TCL) to inhibit the outgrowth of axon-like processes was studied using mouse N2a neuroblastoma cells induced to differentiate by serum withdrawal. At concentrations of 1 and 2 mg/ml (4.97 and 9.94 mM), CB did not cause cell death but inhibited the outgrowth of axon-like processes from N2a cells. This effect was noted as early as 24 h after exposure of the cells to CB. A similar effect was observed with TCL at concentrations of 1 and 2 mg/ml (3.89 and 7.78 mM). Western blot analysis of cell extracts treated with the pesticides showed decreased cross reactivities with the monoclonal antibody RMd09 compared to control extracts. The results indicate that CB and TCL are both able to inhibit axon development and that this effect is associated with reduced levels of the neurofilament high molecular weight protein subunit (NFH). © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Neurodegenerative effects; Carbaryl; Trichlorphon; N2a cells; Neurofilaments
1. Introduction Carbamate and organophosphate (OP) pesticides owe their acute toxicity mainly to the accumulation of acetylcholine following inhibition of neuronal acetylcholinesterase. In addition, these compounds have a number of neurotoxic effects which are not mediated by cholinergic mecha* Corresponding author. Tel.: +44-115-9418-418; fax: + 44-115-948-6636. E-mail address: [email protected] (A.J. Hargreaves)
nisms (Karczmar, 1984; Marquis, 1985). Carbaryl (CB) is the most widely used carbamate pesticide (Hassall, 1990). Although considered as relatively safe, a range of toxic effects have been reported (Cranmer, 1986). In man, clinical evidence suggests that CB neurotoxicity might be occurring more frequently than previously suspected (Branch and Jacqz, 1986). Delayed neurotoxic effects have also been noted following ingestion of an acute dose (Dickoff et al., 1987). Long-term exposure to high levels of CB in the environment has also been found to produce severe, persistent delayed neuropathy (Branch and Jacqz, 1986). In
0378-4274/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 4 2 7 4 ( 9 9 ) 0 0 1 4 2 - 3
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J. Flaskos et al. / Toxicology Letters 110 (1999) 79–84
animals, a single subcutaneous dose can cause paralysis within 24 h which may persist for up to 24 days (Gaines, 1969). Moreover, oral administration of CB daily for 30 days produces mild ataxia (Fisher and Metcalf, 1983). In this work, the neuropathic potential of CB has been investigated further, by studying its neurodegenerative effects directly on cultured nerve cells in vitro. The financial, scientific and ethical considerations favouring the use of cell cultures in neurotoxicology have been duly emphasized (Veronesi, 1992; Atterwill et al., 1994). The mouse N2a neuroblastoma cell line has been employed because it is one of a number of cell lines used regularly in neurotoxicity and pesticide research both for mechanistic and screening purposes (Veronesi, 1992). In addition, it has been sought to establish the nature of the biochemical changes underlying the observed neurodegenerative effects. A widely used OP pesticide, trichlorphon (TCL) has also been studied; this pesticide has an acute in vivo acute toxicity similar to CB and may also cause delayed neuropathy (Hayes, 1982; Abou-Donia and Lapadula, 1990). The results indicate that both carbaryl and trichlorfon reduce the outgrowth of axon-like processes by differentiating N2a cells, an effect which is associated with a reduction in the levels of the neurofilament heavy chain polypeptide.
2. Materials and methods
2.1. Cell lines and reagents Carbaryl (99%) was purchased from Riedel de Hae`n, Seelze, Germany. Trichlorphon (2,2,2trichloro-1-hydroxyethyl phosphonic acid dimethyl ester) and cell culture reagents were obtained from Sigma (Poole, UK). Mouse N2a neuroblastoma and rat C6 glioma cell lines were purchased from ICN (Thane, UK).
2.2. Cell culture conditions The cells were grown and maintained in flasks in Dulbecco’s modified Eagle’s medium (DMEM)
containing 10% fetal calf serum, 1 mM glutamine, penicillin G (100 U/ml) and streptomycin (100 mg/ml). All incubations were performed at 37°C in a humidified environment of 5% CO2 –95% air. The cells were maintained in the logarithmic phase of growth and were subcultured at 4-day intervals.
2.3. Outgrowth of axon-like processes The cells were seeded in 24-well culture dishes, at an initial density of 50 000 cells/ml in 0.5 ml growth medium and incubated for 20 h prior to the induction of cell differentiation by the replacement of growth medium by serum-free medium containing 0.3 mM dibutyryl cAMP (Shea et al., 1991). They were then incubated for 24 or 48 h in the presence and absence of CB or TCL, previously diluted in ethanol, to yield final concentrations of 1 and 2 mg/ml. The final ethanol concentration in the medium of pesticide treated and untreated control cells was 0.5% v/v. In control experiments to determine the selectivity of these effects for cells of neuronal origin, rat C6 glioma cells were induced to differentiate by the addition of 2 mM sodium butyrate. All doses of pesticide used were non-cytotoxic towards the differentiating cells under the conditions employed, as determined by Trypan blue exclusion (Bhuyan et al., 1976) and the reduction of methyl blue tetrazolium (Denizot and Lang, 1986). At the end of the incubation period, the cells were washed with Tris-buffered saline (TBS; 10 mM Tris, 140 mM NaCl, pH 7.4) and fixed for 15 min at −20°C in a solution containing 90% methanol in TBS. After washing, the cells were stained for 1 min at room temperature with Coomassie brilliant blue, washed with TBS and water and then viewed with an inverted light microscope. In experiments using differentiated N2a cells, five random fields were examined in each well and the total number of cells as well as the number of axon-like processes (defined as extensions greater than two cell body diameters in length with an extension foot; Keilbaugh et al., 1991) were recorded, as described by Flaskos et al. (Flaskos et al., 1994, 1998). In experiments using differenti-
J. Flaskos et al. / Toxicology Letters 110 (1999) 79–84
ated C6 cells, the total number of cells and cellular extensions (all of which were greater than two cell body diameters in length) were recorded in five random fields per well.
2.4. Western blot analysis For immunoblot analysis the N2a cells were seeded in a volume of 10 ml of growth medium in culture flasks at a density of 50 000 cells/ml and induced to differentiate as described above. At the end of incubation with pesticides, the cell monolayers were washed with TBS and lysed in electrophoresis sample buffer. The resultant cell lysates were then subjected to gel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE) employing a 7.5% polyacrylamide resolving gel overlaid with a 4% stacking gel (Laemmli, 1970). Equal amounts of cell protein were loaded into each well of the stacking gel. Following electrophoresis, the separated proteins were electrophoretically transferred onto nitrocellulose membrane filters (Towbin et al., 1979). The Western blots obtained were initially blocked with 3% w/v bovine serum albumin in TBS (BSA/TBS) and then incubated overnight at 4°C with the monoclonal antibody RMd09 (culture supernatant diluted 1:200 in BSA/TBS; a gift from Dr M.J. Carden). After six 10-min washes with 0.05% v/v Tween 20 in TBS, the membranes were incubated for 3 h at room temperature with alkaline phosphatase-conjugated rabbit anti-mouse Ig (diluted 1:1000 in BSA/TBS; obtained from Dako, Slough, UK). After six further washings, antibody binding was visualized by incubation of the blots with the alkaline phosphatase substrates 5-bromo-4-chloro-3 indolyl phosphate and nitroblue tetrazolium.
2.5. Statistical analysis Axon outgrowth in the presence of pesticides was expressed as a percentage of control values9 S.E.M. The degree of statistical significance was established using the Mann – Whitney U-test.
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3. Results The neurotoxic potential of CB and TCL was assessed by determining the ability of these drugs to inhibit the extension of axon-like processes from cultured N2a cells under differentiation-promoting conditions. CB and TCL, at doses of 1 and 2 mg/ml (which were previously shown not to be cytotoxic), both decreased significantly (P B 0.05) the number of axon-like extensions 24 h after the induction of cell differentiation and addition of pesticides (Fig. 1 and Table 1). In all treatments employed, the reduction induced by CB was not significantly different from that induced by TCL. The number of axon-like processes formed in the presence of 1 mg/ml CB or TCL after 48 h was not significantly different from that noted after 24 h. By contrast, neither compound inhibited the outgrowth of cellular processes by C6 cells induced to differentiate by the addition of sodium butyrate (data not shown). The nature of the biochemical changes which underlie the above morphological neurotoxic effects was then investigated by probing Western blots of extracts of N2a cells cultured under the above conditions with monoclonal antibody RMd09. This antibody recognizes a non-phosphorylation-dependent epitope on the neurofilament high molecular weight (200 kDa) protein subunit (NFH) (Lee et al., 1988). As it can be seen in Fig. 2, both CB-treated and TCL-treated cell extracts exhibited decreased cross-reactivities with the above antibody when compared to control extracts. The decreased cross-reactivities were noted at both pesticide concentrations and both incubation times.
4. Discussion This work demonstrates that doses of the carbamate pesticide CB and the OP pesticide TCL which do not cause cell death can inhibit the outgrowth of axon-like processes from cultured neuronal cells. This effect is manifested as early as 24 h after exposure of the cells to these agents. Inhibition of extension (neurite) outgrowth from neuroblastoma cells is a particularly useful index
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of neurotoxicity (Campbell et al., 1996; Flaskos et al., 1998) and has been employed in the in vitro screening of neuropathic OPs (see below) (Henschler et al., 1992). A similar inhibition of axonlike extensions by subcytotoxic doses of CB and TCL in the PC12 cell line has also been noted, also of neuronal origin (Fujita et al., 1989), in-
Table 1 The effects of carbaryl (CB) and trichlorphon (TCL) on axon outgrowth in N2a cellsa Treatment
1 mg/ml: 24 h 1 mg/ml: 48 h 2 mg/ml: 24 h
Axon growth (% control) TCL
CB
64.4 914.6 69.2 94.5 56.5 98.4
68.1 9 3.4 77.1 9 4.8 57.4 96.6
a
N2a cells were induced to differentiate in the absence or presence of pesticides. The extent of axon outgrowth was measured as described in Section 2. The results are expressed as a mean percentage of control values 9 S.E.M. All values were significantly different from controls, as determined by the Mann–Whitney U test (PB0.05).
Fig. 2. Western blotting analysis of N2a cell extracts. Cells were induced to differentiate for 24 h in the absence (A) or presence of 1 mg/ml carbaryl (CB) (B) or 1 mg/ml trichlorphon (TCL) (C). Western blots of cell extracts were probed with the monoclonal antibody RMd09. Note the reduction in the crossreactivity of RMd09 with neurofilament high molecular weight protein subunit (NFH) in pesticide-treated cell extracts (arrow).
Fig. 1. Light microscopy of N2a cells induced to differentiate in the absence and presence of carbaryl (CB) or trichlorphon (TCL). Shown are light micrographs of cells fixed and stained with Coomassie brilliant blue after exposure to: no pesticide (A); 1 mg/ml CB (B); 1 mg/ml TCL (C). Note the reduction in axon-like extensions after treatment with pesticides.
duced to differentiate under conditions different to those used in N2a cells (unpublished data). The inability of both compounds to inhibit the outgrowth of processes in C6 cells suggests that these agents exhibit selective effects on the outgrowth of axon-like processes in neuronal cells; a similar result was obtained in previous work using tricresyl phosphate (Flaskos et al., 1998). McLean et al. (1998) observed a similar neurite inhibitory effect
J. Flaskos et al. / Toxicology Letters 110 (1999) 79–84
on the same cell line treated with benomyl; however, in this case, the observed effect was attributed to microtubule disruption. A number of OPs, including TCL, can induce in man and/or other susceptible animals a characteristic delayed neuropathy (OPIDN), in which clinical and morphological signs of axonal degeneration develop at least one week after OP exposure (Johnson, 1975; Abou-Donia and Lapadula, 1990; Lotti, 1992). Several carbamates, under certain conditions, may also produce neuropathy in vivo (Gaines, 1969; Fisher and Metcalf, 1983). With respect to CB, neuropathic effects have been noted in experimental animals following high and/ or repeated doses and under conditions which protect the animals from the acute, cholinergic effects of the drug (Gaines, 1969; Fisher and Metcalf, 1983). In man, clinical evidence suggests that sustained exposure to high doses of CB can cause a severe, persistent delayed neuropathy (Branch and Jacqz, 1986). The relationship between the neuropathy induced by CB to OPIDN is, at present, unclear. Hollinghaus and Fukuto have suggested that carbamate-induced neuropathy is both morphologically and biochemically distinct from OPIDN (Hollinghaus and Fukuto, 1982). On the other hand, Fisher and Metcalf do not preclude the possibility of a common site of action shared by neurotoxic carbamates and OPs (Fisher and Metcalf, 1983). Resolution of these issues depends on the elucidation of the precise biochemical mode of action of these two classes of compounds. Although the molecular basis of OPIDN remains largely unresolved, early events leading to the subsequent manifestation of clinical signs include the disruption of the neurofilament network of the neuronal cytoskeleton (Abou-Donia and Lapadula, 1990; Abou-Donia, 1993). By contrast, little is known about the biochemical mechanisms underlying carbamate neurotoxicity. In view of the large structural diversity of carbamates, it is possible that more than one mechanism is involved. With respect to CB, it has been demonstrated that the early inhibition of axon-like processes is temporally associated with diminished levels of NFH, as determined by decreased cross-reactivity with the monoclonal antibody RMd09 on Western blots.
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Similar effects have also been observed with TCL. Interestingly, a considerable reduction in the levels of NFH, concomitant with reduced axon outgrowth, has also been shown in N2a cells treated with tricresyl phosphate (Flaskos et al., 1998), an OP compound with well established neuropathic potential (Johnson, 1975; Abou-Donia and Lapadula, 1990). The neuronal cytoskeleton plays an important role in the process of neurite development (Cambray-Deakin, 1991) and neurofilaments play a major role in the control of axon girth and stability (Bershadsky and Vasiliev, 1988). The observed decrease in NFH may represent an early biochemical lesion responsible for the inhibitory effects of CB and TCL on neurite development, and may thus be involved in the aetiology of CBand TCL-induced neuropathy in vivo. Alternatively, it may merely reflect the decrease in axon outgrowth and be the consequence of other important lesions caused by these compounds. Further studies are in progress in the laboratory to address these issues and define the relationship of CB-induced neuropathy to OPIDN.
Acknowledgements This work was supported by the Wellcome Trust (grant reference no. 044388), the Commission of the European Communities, the European Science Foundation and the Nuffield Foundation.
References Abou-Donia, M.B., 1993. The cytoskeleton as a target for organophosphorus ester-induced delayed neurotoxicity (OPIDN). Chem.-Biol. Interact. 87, 383 – 393. Abou-Donia, M.B., Lapadula, D.M., 1990. Mechanisms of organophosphorus ester-induced delayed neurotoxicity: type I and type II. Ann. Rev. Pharmacol. Toxicol. 30, 405 – 440. Atterwill, C.K., Bruinink, A., Drejer, J., Duarte, E., Abdulla, E.M., Meredith, C., Nicotera, P., Regan, C., RodriguezFarre´, E., Simpson, M.G., Smith, R., Veronesi, B., Vijverberg, H., Walum, E., Williams, D.C., 1994. In vitro neurotoxicity testing. The report and recommendations of ECVAM workshop 3. ATLA 22, 350 – 362. Bershadsky, A.D., Vasiliev, J.M., 1988. Cytoskeleton. Plenum Press, New York.
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Bhuyan, B.K., Loughman, B.E., Fraser, T.G., Day, K.J., 1976. Comparison of different methods of determining cell viability after exposure to cytotoxic compounds. Exp. Cell Res. 97, 274 – 280. Branch, R.A., Jacqz, E., 1986. Subacute neurotoxicity following long-term exposure to carbaryl. Am. J. Med. 80, 741 – 745. Cambray-Deakin, M.A., 1991. Cytoskeleton of the growing axon. In: Burgoyne, R.D. (Ed.), The Neuronal Cytoskeleton. Wiley-Liss, New York, pp. 233–255. Campbell, I.C., Fletcher, L., Grant, P.A.A., Abdulla, E.M., 1996. Validation of the in vitro tests in neurotoxicology. ATLA 24, 339 – 347. Cranmer, M.F., 1986. Carbaryl-A toxicological review and risk analysis. Neurotoxicology 10, 247–332. Denizot, F., Lang, R., 1986. Rapid colorimetric assay for cell growth and survival — modification to the tetrazolium dye procedure giving improved sensitivity and reliability. J. Immunol. Methods 89, 271–277. Dickoff, D.J., Gerber, O., Turowsky, Z., 1987. Delayed neurotoxicity after ingestion of carbamate pesticide. Neurology 37, 1229 – 1231. Fisher, S.W., Metcalf, R.L., 1983. Production of delayed ataxia by carbamate esters. Pestic. Biochem. Physiol. 19, 243 – 253. Flaskos, J., McLean, W.G., Hargreaves, A.J., 1994. The toxicity of organophosphate compounds towards cultured PC12 cells. Toxicol. Lett. 70, 71–76. Flaskos, J., McLean, W.G., Fowler, M.J., Hargreaves, A.J., 1998. Tricresyl phosphate inhibits the formation of axonlike processes and disrupts neurofilaments in cultured mouse N2a and PC12 cells. Neurosci. Lett. 242, 101–104. Fujita, K., Lazarovici, P., Guroff, G., 1989. Regulation of the differentiation of PC12 pheochromocytoma cells. Environ. Health Perspect. 80, 127–142. Gaines, T.B., 1969. Acute toxicity of pesticides. Toxicol. Appl. Pharmacol. 14, 515 – 534. Hassall, K.A., 1990. The Biochemistry and Uses of Pesticides. Macmillan Press, London. Hayes, W.J. Jr, 1982. Pesticides Studied in Man. Williams and Wilkins, Baltimore. Henschler, D., Schmuck, G., Van Aerssen, M., Schiffmann, D., 1992. The inhibitory effect of neuropathic organophos-
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phate esters on neurite outgrowth in cell cultures: a basis for screening for delayed neurotoxicity. Toxicol. In Vitro 6, 327 – 335. Hollinghaus, J.G., Fukuto, T.R., 1982. The effect of chronic exposure to pesticides on delayed neurotoxicity. J. Agric. Food Chem. 30, 593 – 602. Johnson, M.K., 1975. Organophosphorus esters causing delayed neurotoxic effects. Arch. Toxicol. 34, 259 – 288. Karczmar, A.G., 1984. Acute and long lasting central actions of organophosphorus agents. Fundam. Appl. Toxicol. 4, S1 – S17. Keilbaugh, S.A., Prusoff, W.H., Simpson, M.V., 1991. The PC12 cell as a model for studies of the mechanism of induction of peripheral neuropathy by anti-HIV-I dideoxynucleoside analogs. Biochem. Pharmacol. 42, R5 – R8. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 – 685. Lee, V.M.Y., Otvos, L. Jr, Carden, M.J., Hollosi, M., Dietzchold, B., Lazzarini, R.A., 1988. Identification of the major multiphosphorylation site in mammalian neurofilaments. Proc. Natl. Acad. Sci. USA 85, 1998 – 2002. Lotti, M., 1992. The pathogenesis of organophosphate polyneuropathy. Toxicology 21, 465 – 487. Marquis, J.K., 1985. Noncholinergic mechanisms of insecticide toxicity. TIPS 6, 59 – 60. McLean, W.G., Holme, A.D., Janneh, O, Southgate, G., Howard, C.V., Reed, M.G., 1998. The effect of benomyl on neurite outgrowth in mouse NB2a and human SHSY5Y neuroblastoma cells in vitro. Neurotoxicology 19 (4 – 5), 628 – 632. Shea, T.B., Beermann, M.L., Nixon, R.A., 1991. Multiple proteases regulate neurite outgrowth in NB2a/dl neuroblastoma cells. J. Neurochem. 56, 842 – 851. Towbin, H., Staehelin, T., Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350 – 4354. Veronesi, B., 1992. In vitro screening batteries for neurotoxicants. Neurotoxicology 13, 185 – 196.
The effects of carbaryl and trichlorphon on differentiating mouse N2a neuroblastoma cells John Flaskos a, Maxine J. Fowler b, Cedric Teurtrie b, Alan J. Hargreaves b,* a
Laboratory of Biochemistry and Toxicology, Faculty of Veterinary Medicine, Aristotelian Uni6ersity of Thessaloniki, Thessaloniki, Greece b Department of Life Sciences, Nottingham Trent Uni6ersity, Clifton Lane, Nottingham NG11 8NS, UK Received 22 September 1998; received in revised form 25 July 1999; accepted 27 July 1999
Abstract The ability of the carbamate pesticide carbaryl (CB) and the organophosphate pesticide trichlorphon (TCL) to inhibit the outgrowth of axon-like processes was studied using mouse N2a neuroblastoma cells induced to differentiate by serum withdrawal. At concentrations of 1 and 2 mg/ml (4.97 and 9.94 mM), CB did not cause cell death but inhibited the outgrowth of axon-like processes from N2a cells. This effect was noted as early as 24 h after exposure of the cells to CB. A similar effect was observed with TCL at concentrations of 1 and 2 mg/ml (3.89 and 7.78 mM). Western blot analysis of cell extracts treated with the pesticides showed decreased cross reactivities with the monoclonal antibody RMd09 compared to control extracts. The results indicate that CB and TCL are both able to inhibit axon development and that this effect is associated with reduced levels of the neurofilament high molecular weight protein subunit (NFH). © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Neurodegenerative effects; Carbaryl; Trichlorphon; N2a cells; Neurofilaments
1. Introduction Carbamate and organophosphate (OP) pesticides owe their acute toxicity mainly to the accumulation of acetylcholine following inhibition of neuronal acetylcholinesterase. In addition, these compounds have a number of neurotoxic effects which are not mediated by cholinergic mecha* Corresponding author. Tel.: +44-115-9418-418; fax: + 44-115-948-6636. E-mail address: [email protected] (A.J. Hargreaves)
nisms (Karczmar, 1984; Marquis, 1985). Carbaryl (CB) is the most widely used carbamate pesticide (Hassall, 1990). Although considered as relatively safe, a range of toxic effects have been reported (Cranmer, 1986). In man, clinical evidence suggests that CB neurotoxicity might be occurring more frequently than previously suspected (Branch and Jacqz, 1986). Delayed neurotoxic effects have also been noted following ingestion of an acute dose (Dickoff et al., 1987). Long-term exposure to high levels of CB in the environment has also been found to produce severe, persistent delayed neuropathy (Branch and Jacqz, 1986). In
0378-4274/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 4 2 7 4 ( 9 9 ) 0 0 1 4 2 - 3
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J. Flaskos et al. / Toxicology Letters 110 (1999) 79–84
animals, a single subcutaneous dose can cause paralysis within 24 h which may persist for up to 24 days (Gaines, 1969). Moreover, oral administration of CB daily for 30 days produces mild ataxia (Fisher and Metcalf, 1983). In this work, the neuropathic potential of CB has been investigated further, by studying its neurodegenerative effects directly on cultured nerve cells in vitro. The financial, scientific and ethical considerations favouring the use of cell cultures in neurotoxicology have been duly emphasized (Veronesi, 1992; Atterwill et al., 1994). The mouse N2a neuroblastoma cell line has been employed because it is one of a number of cell lines used regularly in neurotoxicity and pesticide research both for mechanistic and screening purposes (Veronesi, 1992). In addition, it has been sought to establish the nature of the biochemical changes underlying the observed neurodegenerative effects. A widely used OP pesticide, trichlorphon (TCL) has also been studied; this pesticide has an acute in vivo acute toxicity similar to CB and may also cause delayed neuropathy (Hayes, 1982; Abou-Donia and Lapadula, 1990). The results indicate that both carbaryl and trichlorfon reduce the outgrowth of axon-like processes by differentiating N2a cells, an effect which is associated with a reduction in the levels of the neurofilament heavy chain polypeptide.
2. Materials and methods
2.1. Cell lines and reagents Carbaryl (99%) was purchased from Riedel de Hae`n, Seelze, Germany. Trichlorphon (2,2,2trichloro-1-hydroxyethyl phosphonic acid dimethyl ester) and cell culture reagents were obtained from Sigma (Poole, UK). Mouse N2a neuroblastoma and rat C6 glioma cell lines were purchased from ICN (Thane, UK).
2.2. Cell culture conditions The cells were grown and maintained in flasks in Dulbecco’s modified Eagle’s medium (DMEM)
containing 10% fetal calf serum, 1 mM glutamine, penicillin G (100 U/ml) and streptomycin (100 mg/ml). All incubations were performed at 37°C in a humidified environment of 5% CO2 –95% air. The cells were maintained in the logarithmic phase of growth and were subcultured at 4-day intervals.
2.3. Outgrowth of axon-like processes The cells were seeded in 24-well culture dishes, at an initial density of 50 000 cells/ml in 0.5 ml growth medium and incubated for 20 h prior to the induction of cell differentiation by the replacement of growth medium by serum-free medium containing 0.3 mM dibutyryl cAMP (Shea et al., 1991). They were then incubated for 24 or 48 h in the presence and absence of CB or TCL, previously diluted in ethanol, to yield final concentrations of 1 and 2 mg/ml. The final ethanol concentration in the medium of pesticide treated and untreated control cells was 0.5% v/v. In control experiments to determine the selectivity of these effects for cells of neuronal origin, rat C6 glioma cells were induced to differentiate by the addition of 2 mM sodium butyrate. All doses of pesticide used were non-cytotoxic towards the differentiating cells under the conditions employed, as determined by Trypan blue exclusion (Bhuyan et al., 1976) and the reduction of methyl blue tetrazolium (Denizot and Lang, 1986). At the end of the incubation period, the cells were washed with Tris-buffered saline (TBS; 10 mM Tris, 140 mM NaCl, pH 7.4) and fixed for 15 min at −20°C in a solution containing 90% methanol in TBS. After washing, the cells were stained for 1 min at room temperature with Coomassie brilliant blue, washed with TBS and water and then viewed with an inverted light microscope. In experiments using differentiated N2a cells, five random fields were examined in each well and the total number of cells as well as the number of axon-like processes (defined as extensions greater than two cell body diameters in length with an extension foot; Keilbaugh et al., 1991) were recorded, as described by Flaskos et al. (Flaskos et al., 1994, 1998). In experiments using differenti-
J. Flaskos et al. / Toxicology Letters 110 (1999) 79–84
ated C6 cells, the total number of cells and cellular extensions (all of which were greater than two cell body diameters in length) were recorded in five random fields per well.
2.4. Western blot analysis For immunoblot analysis the N2a cells were seeded in a volume of 10 ml of growth medium in culture flasks at a density of 50 000 cells/ml and induced to differentiate as described above. At the end of incubation with pesticides, the cell monolayers were washed with TBS and lysed in electrophoresis sample buffer. The resultant cell lysates were then subjected to gel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE) employing a 7.5% polyacrylamide resolving gel overlaid with a 4% stacking gel (Laemmli, 1970). Equal amounts of cell protein were loaded into each well of the stacking gel. Following electrophoresis, the separated proteins were electrophoretically transferred onto nitrocellulose membrane filters (Towbin et al., 1979). The Western blots obtained were initially blocked with 3% w/v bovine serum albumin in TBS (BSA/TBS) and then incubated overnight at 4°C with the monoclonal antibody RMd09 (culture supernatant diluted 1:200 in BSA/TBS; a gift from Dr M.J. Carden). After six 10-min washes with 0.05% v/v Tween 20 in TBS, the membranes were incubated for 3 h at room temperature with alkaline phosphatase-conjugated rabbit anti-mouse Ig (diluted 1:1000 in BSA/TBS; obtained from Dako, Slough, UK). After six further washings, antibody binding was visualized by incubation of the blots with the alkaline phosphatase substrates 5-bromo-4-chloro-3 indolyl phosphate and nitroblue tetrazolium.
2.5. Statistical analysis Axon outgrowth in the presence of pesticides was expressed as a percentage of control values9 S.E.M. The degree of statistical significance was established using the Mann – Whitney U-test.
81
3. Results The neurotoxic potential of CB and TCL was assessed by determining the ability of these drugs to inhibit the extension of axon-like processes from cultured N2a cells under differentiation-promoting conditions. CB and TCL, at doses of 1 and 2 mg/ml (which were previously shown not to be cytotoxic), both decreased significantly (P B 0.05) the number of axon-like extensions 24 h after the induction of cell differentiation and addition of pesticides (Fig. 1 and Table 1). In all treatments employed, the reduction induced by CB was not significantly different from that induced by TCL. The number of axon-like processes formed in the presence of 1 mg/ml CB or TCL after 48 h was not significantly different from that noted after 24 h. By contrast, neither compound inhibited the outgrowth of cellular processes by C6 cells induced to differentiate by the addition of sodium butyrate (data not shown). The nature of the biochemical changes which underlie the above morphological neurotoxic effects was then investigated by probing Western blots of extracts of N2a cells cultured under the above conditions with monoclonal antibody RMd09. This antibody recognizes a non-phosphorylation-dependent epitope on the neurofilament high molecular weight (200 kDa) protein subunit (NFH) (Lee et al., 1988). As it can be seen in Fig. 2, both CB-treated and TCL-treated cell extracts exhibited decreased cross-reactivities with the above antibody when compared to control extracts. The decreased cross-reactivities were noted at both pesticide concentrations and both incubation times.
4. Discussion This work demonstrates that doses of the carbamate pesticide CB and the OP pesticide TCL which do not cause cell death can inhibit the outgrowth of axon-like processes from cultured neuronal cells. This effect is manifested as early as 24 h after exposure of the cells to these agents. Inhibition of extension (neurite) outgrowth from neuroblastoma cells is a particularly useful index
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of neurotoxicity (Campbell et al., 1996; Flaskos et al., 1998) and has been employed in the in vitro screening of neuropathic OPs (see below) (Henschler et al., 1992). A similar inhibition of axonlike extensions by subcytotoxic doses of CB and TCL in the PC12 cell line has also been noted, also of neuronal origin (Fujita et al., 1989), in-
Table 1 The effects of carbaryl (CB) and trichlorphon (TCL) on axon outgrowth in N2a cellsa Treatment
1 mg/ml: 24 h 1 mg/ml: 48 h 2 mg/ml: 24 h
Axon growth (% control) TCL
CB
64.4 914.6 69.2 94.5 56.5 98.4
68.1 9 3.4 77.1 9 4.8 57.4 96.6
a
N2a cells were induced to differentiate in the absence or presence of pesticides. The extent of axon outgrowth was measured as described in Section 2. The results are expressed as a mean percentage of control values 9 S.E.M. All values were significantly different from controls, as determined by the Mann–Whitney U test (PB0.05).
Fig. 2. Western blotting analysis of N2a cell extracts. Cells were induced to differentiate for 24 h in the absence (A) or presence of 1 mg/ml carbaryl (CB) (B) or 1 mg/ml trichlorphon (TCL) (C). Western blots of cell extracts were probed with the monoclonal antibody RMd09. Note the reduction in the crossreactivity of RMd09 with neurofilament high molecular weight protein subunit (NFH) in pesticide-treated cell extracts (arrow).
Fig. 1. Light microscopy of N2a cells induced to differentiate in the absence and presence of carbaryl (CB) or trichlorphon (TCL). Shown are light micrographs of cells fixed and stained with Coomassie brilliant blue after exposure to: no pesticide (A); 1 mg/ml CB (B); 1 mg/ml TCL (C). Note the reduction in axon-like extensions after treatment with pesticides.
duced to differentiate under conditions different to those used in N2a cells (unpublished data). The inability of both compounds to inhibit the outgrowth of processes in C6 cells suggests that these agents exhibit selective effects on the outgrowth of axon-like processes in neuronal cells; a similar result was obtained in previous work using tricresyl phosphate (Flaskos et al., 1998). McLean et al. (1998) observed a similar neurite inhibitory effect
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on the same cell line treated with benomyl; however, in this case, the observed effect was attributed to microtubule disruption. A number of OPs, including TCL, can induce in man and/or other susceptible animals a characteristic delayed neuropathy (OPIDN), in which clinical and morphological signs of axonal degeneration develop at least one week after OP exposure (Johnson, 1975; Abou-Donia and Lapadula, 1990; Lotti, 1992). Several carbamates, under certain conditions, may also produce neuropathy in vivo (Gaines, 1969; Fisher and Metcalf, 1983). With respect to CB, neuropathic effects have been noted in experimental animals following high and/ or repeated doses and under conditions which protect the animals from the acute, cholinergic effects of the drug (Gaines, 1969; Fisher and Metcalf, 1983). In man, clinical evidence suggests that sustained exposure to high doses of CB can cause a severe, persistent delayed neuropathy (Branch and Jacqz, 1986). The relationship between the neuropathy induced by CB to OPIDN is, at present, unclear. Hollinghaus and Fukuto have suggested that carbamate-induced neuropathy is both morphologically and biochemically distinct from OPIDN (Hollinghaus and Fukuto, 1982). On the other hand, Fisher and Metcalf do not preclude the possibility of a common site of action shared by neurotoxic carbamates and OPs (Fisher and Metcalf, 1983). Resolution of these issues depends on the elucidation of the precise biochemical mode of action of these two classes of compounds. Although the molecular basis of OPIDN remains largely unresolved, early events leading to the subsequent manifestation of clinical signs include the disruption of the neurofilament network of the neuronal cytoskeleton (Abou-Donia and Lapadula, 1990; Abou-Donia, 1993). By contrast, little is known about the biochemical mechanisms underlying carbamate neurotoxicity. In view of the large structural diversity of carbamates, it is possible that more than one mechanism is involved. With respect to CB, it has been demonstrated that the early inhibition of axon-like processes is temporally associated with diminished levels of NFH, as determined by decreased cross-reactivity with the monoclonal antibody RMd09 on Western blots.
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Similar effects have also been observed with TCL. Interestingly, a considerable reduction in the levels of NFH, concomitant with reduced axon outgrowth, has also been shown in N2a cells treated with tricresyl phosphate (Flaskos et al., 1998), an OP compound with well established neuropathic potential (Johnson, 1975; Abou-Donia and Lapadula, 1990). The neuronal cytoskeleton plays an important role in the process of neurite development (Cambray-Deakin, 1991) and neurofilaments play a major role in the control of axon girth and stability (Bershadsky and Vasiliev, 1988). The observed decrease in NFH may represent an early biochemical lesion responsible for the inhibitory effects of CB and TCL on neurite development, and may thus be involved in the aetiology of CBand TCL-induced neuropathy in vivo. Alternatively, it may merely reflect the decrease in axon outgrowth and be the consequence of other important lesions caused by these compounds. Further studies are in progress in the laboratory to address these issues and define the relationship of CB-induced neuropathy to OPIDN.
Acknowledgements This work was supported by the Wellcome Trust (grant reference no. 044388), the Commission of the European Communities, the European Science Foundation and the Nuffield Foundation.
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