2,4-Dichlorophenoxyacetic Acid Disrupts the Cytoskeleton and Disorganizes the Golgi Apparatus of Cultured Neurons

Silvana B. Rosso*,1, Alfredo O. Cáceres{dagger}, Ana Maria Evangelista de Duffard*, Ricardo O. Duffard* and Santiago Quiroga{ddagger},2

* Laboratorio de Toxicología Experimental, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina; {dagger} Instituto de Investigación Médica Mercedes y Martín Ferreyra (INIMEC-CONICET), Córdoba, Argentina; and {ddagger} Departamento de Química Biológica-CIQUIBIC, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba-CONICET, Argentina

Received January 28, 2000; accepted April 5, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
2,4-Dichlorophenoxyacetic acid (2,4-D) is a potent neurotoxic herbicide widely used in agriculture. The basic mechanisms by which 2,4-D produces cell damage have not yet been determined. In this study we have examined the effects of 2,4-D in primary cultures of cerebellar granule cells in order to obtain insights into the possible mechanisms underlying the toxic effects of this herbicide. The results obtained indicate that a 24-hour exposure to 2,4-D produces a striking and dose-dependent inhibition of neurite extension. This phenomenon is paralleled by a significant reduction in the cellular content of both dynamic and stable microtubules, a disorganization of the Golgi apparatus, and an inhibition in the synthesis of complex gangliosides. Interestingly, 2,4-D inhibits the in vitro polymerization of purified tubulin. Taken together, the present observations raise the possibility that at least one basic mechanism underlying 2,4-D neurotoxicity involves an inhibition of microtubule assembly. That event may cause a decreased neurite outgrowth response, and could also explain the observed differences in the pattern of ganglioside biosynthesis and/or the disorganization of the Golgi apparatus.

Key Words: 2,4-D; neurotoxicity; developing neurons; microtubules; gangliosides; Golgi apparatus.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
2–4-Dichlorophenoxyacetic acid (2,4-D) is a potent herbicide widely used around the world in order to eradicate dicotyledonian weeds from crop lands, pastures, and other plantations intended for cultivation of crops for human and animal consumption (Feldman and Maibach, 1974Go; Grover et al., 1986Go). Unfortunately, 2,4-D produces different harmful effects on mammals, including humans, which range from embryotoxicity and teratogenicity to neurotoxicity. For example, during the pre- and post-natal periods, the central nervous system (CNS) appears to be one of the main targets of the toxic effects of phenoxyherbicides such as 2,4-D. Thus, exposure of young animals to this herbicide results in a decrease in the total ganglioside content of the brain and in a reduced expression of complex gangliosides suggestive of a delay in brain development (Rosso et al., 1997Go). In addition, it produces a deficit in myelin lipid's deposition (Duffard et al., 1996Go; Mori de Moro et al., 1985Go; Rosso et al., 2000Go), astrogliosis (Brusco et al., 1997Go) and behavioral alterations (Evangelista et al., 1995Go; Mohammad and Omer, 1986Go; Oliveira and Palermo-Neto, 1993Go; Rosso et al., 2000Go). Retinal degeneration and miotony have also been reported after a long-term exposure to 2,4-D (Argüello et al., 1990Go; Bernard et al., 1985Go; Mattsson et al., 1997Go).

Surprisingly, no efforts have been made in order to determine whether or not the deleterious effects of 2,4-D result from a direct action of the herbicide on developing neurons; in addition, very little is known about the cellular mechanisms underlying its neurotoxicity. With these considerations in mind, in the present study we analyzed the effects of 2,4-D on the development of cultured cerebellar granule cells. The results obtained suggest 2,4-D has a direct toxic effect on these neurons by altering key cellular elements involved in neuronal morphogenesis, namely the microtubular cytoskeleton and the Golgi apparatus.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell cultures.
Cerebellar granule cells were cultured essentially as described by Gallo et al., (1987). Briefly, cerebella of 8 days old rats were dissected aseptically in Hank's balanced salt solution. After removal of the meninges, the cells were dissociated using a low concentration of trypsin followed by trituration. The dissociated cells were plated onto coverslips previously coated with poly-L-lysine (100 µg/ml in 0.1 M borate buffer, pH 8.3) at low density (1 x 105 cells/cm2) for immunofluorescence experiments or at a higher density (3 x 105 cells/cm2) for those involving metabolic labeling. Cells were maintained with DMEM plus 10% fetal calf serum; cultures were placed in a humidified 370C incubator with 5% CO2. When indicated, cultures were treated with different concentrations of 2,4-D (dissolved in 90% ethanol) starting 2 h after plating. The final concentration of ethanol in the culture medium was 0.09%. The control cells were maintained in culture medium containing an equal concentration of ethanol.

DNA quantification.
The DNA content of control and 2,4-D-treated cells was measured using Hoeschst 33258 (Sigma Chemical Co.-concentration 1 µg/ml) as a fluorophore in an SLM-Aminco 4800-C spectrofluorometer (Labarca and Paigen, 1980Go). The DNA content was calculated in each sample by extrapolating the obtained values to a calibration curve made with standard DNA (Sigma Chemical Co., St. Louis, MO).

Determination of cell viability.
The assessment of cell viability was performed as described (Busciglio et al., 1992Go). In brief, living cells, after 24 h of DIV, were incubated with 10 µg/ml propidium iodide (PI) for 30 min., washed with phosphate buffered saline, and fixed as described. Viable neurons exclude PI. A total of 400 cells was scored for each experimental condition.

Determination of gangliosides biosynthesis.
Cerebellar granular cells (control and 2,4-D treated) were cultured for 24 h in the presence of 10 µCi (per dish) 3H-D-galactose (specific activity 34.60 Ci/mmol, New England Nuclear, Boston). The cells were then harvested, washed, and gangliosides extracted with chloroform-methanol-water (60:30:4.5 by volume). After drying, the extracts were re-dissolved in a chloroform-methanol-water solution (3:48:47 by volume) and purified through C18 reversed-phase columns (Williams and McCluer, 1980Go). The purified lipids were resolved by HPTLC on silica gel G (Merck, Germany) using chloroform-methanol-0.25% CaCl2 (60:35:8 by volume). The plates were prepared for fluorography as described (Bonner and Stedman, 1978Go), and the resulting fluorograms scanned in a Shimadzu chromato scanner CS930.

Immunofluorescence.
Cells were fixed prior to or after a mild detergent extraction under microtubule-stabilizing conditions (Nakata and Hirokawa, 1987Go) and processed for immunofluorescence as previously described (Cáceres et al., 1992Go; DiTella et al., 1994Go). The primary antibodies used were: a monoclonal antibody (mAb) against tyrosinated {alpha}-tubulin (clone TUB-1A2, mouse IgG, Sigma Chemical Co., St. Louis, MO) diluted 1:600, a mAb against the neuron-specific class III ß-tubulin isotype (clone TuJ1, mouse IgG) diluted 1:100; and a rabbit polyclonal antibody that recognizes detyrosinated {alpha}-tubulin (Cáceres et al., 1992Go) diluted 1:200. The antibody-staining protocol entailed labeling with the first primary antibody, washing with PBS, staining with labeled secondary antibody (fluorescein- or rhodamine-conjugated), and washing similarly; the same procedure was repeated for the second primary antibody. Incubations with primary antibodies were for 1 or 3 h at room temperature, while incubations with secondary antibodies were performed for 1 h at 37°C. The cells were observed with an inverted microscope (Carl Zeiss Axiovert 35M) equipped with epifluorescence and differential interference contrast (DIC) optics. Fluorescent images were captured under regular fluorescence microscopy with a silicon-intensified target camera (SIT-C2400 Hamamatsu Corp., Middlesex, NJ) or with a Micromax cooled CCD camera (Princeton, NJ). The images were digitized directly into a Metamorph/Metafluor Image Processor (Universal Imaging Corporation, West Chester, PA). Photographs were printed using Adobe Photoshop. To measure neuronal shape parameters, cells were randomly selected and traced from a video screen using the morphometric menu of the Metamorph, as described previously (Cáceres et al., 1992Go; DiTella et al., 1994Go; Feiguin et al., 1994Go; Paglini et al., 1998Go).

Staining with BODIPY FL C5-ceramide.
Control and 2,4-D-treated living granule cells were stained with BODIPY FL C5-ceramide (Molecular Probes, Eugene, OR) for 15 min at 37°C as described (Pagano et al., 1991Go). After washing, the cells were fixed with 4% paraformaldehyde-0.12 M sucrose in PBS for 20 min at room temperature; they were then observed in the inverted microscope and images obtained as described previously (Feiguin et al., 1994Go).

Assays of microtubule polymerization in vitro.
Microtubular proteins (tubulin plus microtubule-associated proteins, e.g., MAPs) were obtained from 15-day-old rat brains as described by Borisy et al. (1975); from this preparation, pure tubulin was obtained using a phosphocellulose column (Weingarten et al., 1975Go). Tubulin polymerization, in the presence or absence of MAPs, was induced with 1 mM GTP and 15 mM taxol (Calbiochem-Novabiochem Corp., La Jolla, CA) under constant agitation at 37°C. Polymerization assays were monitored by light scattering at 350 nm wavelength in a SLM-Aminco 4800 C spectrofluorometer; reactions were performed in a final volume of 0.8 ml containing 0.1 M MES pH 6.9, 1 mM EGTA, 2 mM MgCl2 in the presence or absence of 2,4-D (1 or 2 mM).

Statistical analyses.
Results were analyzed using one-way analysis of variance (ANOVA) followed by the Student Neuman-Keuls multiple comparison test for unequal replications.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cultured cerebellar granule cells have proven to be an excellent model system for studying neurite outgrowth and the expression of cytoskeletal proteins involved in nerve cell morphogenesis (Goold et al., 1999Go; Lucas et al., 1998Go). Under our culture conditions, 90% of the cells had extended neurites one day after plating; typically, these include one axon-like neurite of more than 100 µm in length and 1–2 much shorter and thinner minor processes that will later develop as dendrites. In these cells, both axon-like neurites and minor processes stain intensely with antibodies against tubulin, including the mAb TuJ1 that recognizes the class III neuron-specific ß-tubulin isotype. This rapid neurite outgrowth response offers an excellent opportunity to examine in detail the effects of 2,4-D on neuronal differentiation. Therefore, in the first set of experiments, process formation was quantitated in control and 2,4-D-treated cells, 1 day after plating, by morphometry of fixed cultures. The results obtained show that there is a significant and dose-dependent reduction in the length of axon-like and minor neurites in cells cultured in the presence of 2,4-D, when compared with untreated cells (Table 1Go). By contrast, no changes in the size of the cell body, the nucleus, or the number of neuritic processes were detected between control and 2,4-D-treated cells (Table 1Go). We also studied cell viability of the control and 2,4-D-treated cells using the PI fluorescence method. The results indicate that 2,4-D did not cause any significant loss of neuron viability at any of the doses assayed (Control: 95.2 ± 3.5; 1 mM 2,4-D: 94.8 ± 4.3 % and 2 mM 2,4-D: 93.8 ± 2.8 of viable neurons after 24 h in culture, respectively). Since it is now well established that microtubule assembly and stabilization are essentially required for process formation in developing neurons (Cáceres et al., 1992Go), it became of interest to examine whether 2,4-D has any effect on the distribution and/or content of dynamic and stable microtubules. For such a purpose, control and 2, 4-D-treated cultures, fixed after detergent extraction performed under microtubule-stabilizing conditions, were labeled with antibodies that recognize either dynamic (containing tyrosinated {alpha}-tubulin) or stable (containing detyrosinated {alpha}-tubulin) microtubules. A considerable reduction of {alpha}-tubulin immunolabeling was detected in cells treated with 2,4-D; as expected, this phenomenon occurred in all the neurons displaying a decreased neurite outgrowth response (Figs. 1A–1FGo). Quantitative fluorescence measurements confirmed this observation and clearly revealed that 2,4-D produced a significant reduction of both tyrosinated and detyrosinated {alpha}-tubulin immunolabeling (Table 2Go). The decrease of tyrosinated {alpha}-tubulin immunolabeling was not observed when the cells were extracted with detergents after fixation; by contrast, the reduction of detyrosinated {alpha}-tubulin immunolabeling was observed independent of whether or not the cells were extracted with detergents prior to or after fixation. These observations suggest that 2,4-D is not affecting tubulin expression, but rather altering microtubule assembly and/or stability.


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TABLE 1 Effect of 2,4-D on Neuronal Shape Parameters in Cerebellar Granule Cells after Development in Culture for 24 Hours
 


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FIG. 1. Double-immunofluorescence micrographs showing the distribution of tyrosinated (A, C, E) and detyrosinated (B, D, F) {alpha}-tubulin in cerebellar granule cells from control (A, B) and 2,4-D-treated (C–F) neurons. Cultures were treated with 1 mM (C, D) or 2 mM (E, F) 2,4-D. For this experiment cells were extracted with detergents prior to fixation under microtubule-stabilizing conditions. Note that 2,4-D decreases tubulin immunolabeling and inhibits neurite extension. Calibration bar: 10 µm.

 

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TABLE 2 Fluorescence Intensity Measurements of Tubulin Immunolabeling in Cultured Cerebellar Granule Cells Treated with 2,4-D for 24 Hours
 
To directly test this possibility, the effect of 2,4-D on the polymerization of both pure tubulin and microtubular proteins (containing tubulin and MAPs) was measured by light scattering. Figure 2Go shows that 2,4-D, used at the same concentrations that inhibit neurite extension and reduce the cellular content of dynamic and stable microtubules, has a significant inhibitory effect on the polymerization of microtubular proteins (Fig. 2AGo, polymerization induced by GTP) or pure tubulin (Fig. 2BGo, polymerization induced by taxol). This inhibitory effect ranged from 30% for the 1 mM dose to 50–60% for the 2 mM dose, and it was only observed when 2,4-D was present in the assembly buffer before the addition of either GTP or taxol, but not when the herbicide was added to previously assembled microtubules (not shown). Taken together, these results suggest that, rather than being a microtubule-destabilizing agent, 2,4-D has a direct effect on tubulin assembly that appears to be independent of the presence of MAPs.



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FIG. 2. Effects of 2,4-D (1 mM) on the polymerization rate of total brain microtubular protein (A) and pure tubulin (B). Note that 2,4-D inhibits the polymerization of both total microtubular protein and pure tubulin.

 
In previous studies we have shown that exposure of young animals to 2,4-D is concomitant with significant changes in the pattern of ganglioside biosynthesis in the CNS (Mori de Moro et al., 1985Go; Rosso et al., 1997Go). Since the biosynthesis of the most complex gangliosides depends on the integrity of the Golgi complex (Giraudo et al., 1999Go; Maccioni et al., 1999Go; van Echten and Sandhoff, 1989Go), and since Golgi structures are disrupted after microtubule depolymerization (Karecla and Kreis, 1992Go; Kreis, 1990Go; Scheel et al., 1990Go), it became of interest to examine whether or not exposure of cultured granule cells to 2,4-D has any effect on the organization of the Golgi apparatus and/or in the biosynthesis of gangliosides. For the first type of experiment, cells were cultured for 1 day in the presence or absence of 2,4-D and the Golgi apparatus visualized by labeling with bodipy-ceramide. BODIPY FL C5-ceramide is a fluorescent lipid marker that accumulates at the Golgi complex of living cells in culture; interestingly, the fluorescent emission spectrum of this probe is dramatically red-shifted as it accumulates in the Golgi complex (Pagano et al., 1991Go). As shown in Figure 3AGo, in control cells, bodipy-ceramide labeling is detected predominantly in the cell body, and to a less extent in neurites, when observed with the fluorescein filter. Within the cell body and in more than 90% of the cells, maximum labeling was detected in a single spot located in the immediate vicinity of the cell nucleus. This structure was readily visible and the only one detected when using the rhodamine filter, clearly indicating that it corresponds to the Golgi apparatus (Fig. 3BGo, arrowhead). In neurons treated with 1-mM 2,4-D, bodipy-ceramide also accumulates in a round structure located close to the cell nucleus (Figs. 3C and 3DGo, arrowheads). However, several vesicle-like structures that distribute throughout the cell body were readily visible in both the fluorescein and rhodamine filters (Figs. 3C and 3DGo). A more dramatic effect was observed when cells were treated with 2-mM 2,4-D. Under this condition, all of the staining visible with the rhodamine filter was localized to small vesicle-like structures scattered throughout the cytoplasm (Figs. 3F and 3HGo).



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FIG. 3. 2,4-D disorganizes the Golgi apparatus of cerebellar granular neurons in culture. Micrographs showing cerebellar granule cells from control cultures (A and B) and from similar ones treated with 1 mM (C, D) or 2 mM (E-H) 2,4-D. For this experiments the cells were cultured for 24 h, after that time period they were stained with BODIPY FL C5-ceramide and observed in the fluorescence microscope using the fluorescein (A, C, E, and G) and rhodamine (B, D, F, and H) filters. Note the dramatic fragmentation of the Golgi complex in neurons exposed to 2,4-D. Calibration bar: 10 µm.

 
We then analyzed the total rate of ganglioside biosynthesis by labeling cultured cerebellar granular neurons with 3H-galactose. The results (expressed as incorporation of 3H-galactose into the ganglioside fraction divided by the total amount of DNA in each culture) indicate that 2,4-D produces a significant reduction of ganglioside biosynthesis at all the concentrations assayed. These inhibition rates were from about 35% for 0.2 mM to about 85% for the 2 mM concentration (Fig. 4Go). These values are in line with those previously reported for the brains of young postnatal rats exposed to 2,4-D (Rosso et al., 1997Go). The pattern of ganglioside composition was also analyzed using the method described above, followed by thin layer chromatography (see Materials and Methods). Analysis of the labeling of individual gangliosides (Table 3Go) showed that in cells treated with concentrations of 2,4-D above 0.5 mM there is a marked reduction in the relative amounts of gangliosides belonging to the gangliotetraosyl series (e.g., GD1b and GT1b). This reduction is concomitant with a striking increase in the relative amount of more simple gangliosides, such as GD3 and GM3. The change from a predominant biosynthesis of GM3 and GD3 to one of gangliosides of the gangliotetraosyl series is a well-established marker of normal neuronal differentiation (Ando, 1983Go), and therefore, our results suggest that 2,4-D directly affects this event. In this regard, it is worth mentioning that similar alterations in the pattern of gangliosides biosynthesis has been observed with other agents that inhibit neuronal differentiation and are toxic for neurons, like the high density lipoprotein (Kivatinitz et al., 1995Go; 1997Go).



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FIG. 4. 2,4-D significantly inhibits the incorporation of 3H-D-galactose into gangliosides. Effects of 2,4-D on the incorporation of 3H-D-galactose into gangliosides in cerebellar granular neurons exposed to different concentration (0.2, 0.5, 1.0, and 2.0 mM) of 2,4-D for 24 h. Control cells were exposed to an equal volume of vehicle (ethanol). Each bar represents the mean ± SEM; n = 6; Asterisks indicate p < 0.001 statistically different from control cells.

 

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TABLE 3 Gangliosides Biosynthesis in Cultured Cerebellar Granule Cells Treated with 2,4-D for 24 Hours
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Our study results provide evidence indicating that 2,4-D has a direct, dose-dependent toxic effect on developing cerebellar granule cells. In these neurons, 2,4-D toxicity is characterized by a significant inhibition of neurite extension after a 24-h exposure to the herbicide. It is unlikely that this phenomenon reflects a general toxic response of neurons to the herbicide, since no alterations in cell body size or nuclear shrinkage were detected in 2,4-D-treated cells. In addition, the number of primary neurites that 2,4-D-treated neurons extended was identical to that of control cells. On the other hand, inhibition of neurite extension in 2,4-D-treated granule cells was accompanied by decreased immunostaining for tyrosinated and detyrosinated {alpha}-tubulin. Since the decrease in tyrosinated {alpha}-tubulin immunolabeling only became evident when the cells were extracted with detergents under microtubule stabilizing conditions prior to fixation, our results suggest that neurons treated with 2,4-D have a decreased content of both dynamic and stable microtubules, and therefore the herbicide may be inhibiting microtubule assembly. In this regard, it is worth mentioning that alterations in the organization of microtubules and microfilaments have also been reported in NIH 3T3 fibroblasts exposed to 2,4-D and 2,4,5-trichlorophenoxyacetic acid (Zhao et al., 1987Go). Our observations also raise the possibility that the inhibitory effect of 2,4-D on microtubule assembly may result from direct interaction of 2,4-D with tubulin. Several lines of evidence favor such a possibility. First, 2,4-D is transported across cellular membranes, probably using the organic anion transporter protein, oatp3 (Bergesse and Balegno, 1995Go; Villalobos et al., 1996Go). In addition, we have evidence showing that 2,4-D is transported into cultured cerebellar granular cells in a dose-dependent, saturable manner (Cáceres, Rosso, Quiroga, unpublished observations). Interestingly, oatp3 is highly expressed by granule cells (Abe et al., 1998Go). Secondly, and perhaps more importantly, we show here that 2,4-D significantly reduces tubulin polymerization in vitro; since this effect occurs independent of the presence of MAPs, a direct interaction of 2,4-D with tubulin is suggested. In this regard, it is important to point out that, in contrast to other microtubule depolymerizing drugs such as nocodazole or colchicine that bind to tubulin, 2,4-D does not disassemble previously polymerized microtubules (not shown). However, as in the case of nocodazole or colchicine, it is likely that 2,4-D inhibits neurite extension by preventing microtubule assembly, an essential requirement for process formation in developing neurons (Cáceres et al., 1992Go; Drubin et al., 1985Go). At present, we cannot discard the possibility that 2,4-D may be causing microtubule depolymerization by other mechanisms in addition to that derived from its direct interaction with tubulin (see above). For example, it has been shown that 2,4-D elevates intracellular calcium concentration in hepatocytes (Palmeira et al., 1995Go) and rapid changes in intracellular calcium occur in cerebellar granule cells following exposure to other toxicants, such as polychlorinated biphenyls (Kodavanti et al., 1993Go), and methyl mercury (Sarafian, 1993Go). It has been also demonstrated that calcium can directly destabilize growing microtubules, most probably by increasing the rate of GTP hydrolysis at the GTP cap (O'Brien et al., 1997Go).

2,4-D also produces a striking fragmentation of the Golgi apparatus, as shown by staining with bodipy-ceramide. As in the case of the inhibition of neurite extension, this fragmentation is probably related to the alterations in microtubule organization observed in 2,4-D-treated neurons. A close structural relationship between the microtubule cytoskeleton and Golgi apparatus is well documented (Feiguin et al., 1994Go; Karecla and Kreis, 1992Go; Kreis, 1990Go; Scheel et al., 1990Go) and microtubule depolymerization has dramatic consequences on the typical perinuclear localization of the Golgi apparatus, with Golgi membranes suffering a reversible fragmentation and dispersal throughout the cytoplasm (Kreis, 1990Go).

Whether or not the alterations in the biosynthesis of gangliosides are a consequence of 2,4-D-induced Golgi disorganization remains an open question. For example, agents that disrupt the Golgi apparatus without affecting microtubules, such as Brefeldin A, inhibit the synthesis of gangliosides belonging to the gangliotetraosyl series (GM1, GD1a, GD1b, GT1b) and produce a significant accumulation of GM3 and GD3 in several cell systems (Giraudo et al., 1999Go; Maccioni et al., 1999Go). On the other hand, agents that cause Golgi fragmentation as a consequence of microtubule depolymerization, such as nocodazole or colchicine, do not produce noticeable changes in the process of intra-Golgi transport (Scheel et al., 1990Go) and on the biosynthesis of complex gangliosides (Ogiso et al., 1992Go). Therefore, our data suggest that 2,4-D may have additional effects on this organelle. However, regardless of the precise mechanism, it is worth noting that the decrease in complex gangliosides observed in 2,4-D-treated neurons may also explain the inhibition of neurite extension. Thus, it has recently been shown that transgenic mice lacking complex gangliosides develop axonal degeneration in the central and the peripheral nervous system, as well as decreased central myelination (Sheikh et al., 1999Go), features that resemble 2,4-D neurotoxicity.

In summary, we propose that the primary toxic effects of 2,4-D on neurons are most probably due to inhibition of microtubule polymerization. The significant reduction of both stable and dynamic microtubules can inhibit the process of neuronal differentiation and neurite extension. At the same time, 2,4-D produces a significant disorganization of the Golgi complex that can also affect neurite formation and may be reflected by changes in the pattern of ganglioside biosynthesis.


    ACKNOWLEDGMENTS
 
The authors wish to thank Dr. Gerardo Fidelio for his collaboration in the in vitro polymerization experiments. SR is a recipient of a CONICET fellowship. This work was supported by grants from: Agencia Nacional de Promoción Científica (PICT) 05–00000–00937 (to AC and SQ) and 05–00001–00012 (to RD), CONICET (to SQ and AC), CONICOR (to SQ) and Secyt-U.N.Cba. (to SQ).


    NOTES
 
1 Present address: Dpto. de Química Biológica, Fac. de Ciencias Químicas, Universidad Nacional de Córdoba, Pabellón Argentina, 5000-Córdoba, Argentina. Back

2 To whom correspondence should be addressed at Dpto. de Química Biológica, Fac. de Ciencias Químicas, Universidad Nacional de Córdoba, Pabellón Argentina, 5000-Córdoba, Argentina. Fax: (54)-(351)-433–4074. E-mail: squiroga{at}dqbfcq.uncor.edu. Back


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 DISCUSSION
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