* Laboratorio de Toxicología Experimental, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina;
Instituto de Investigación Médica Mercedes y Martín Ferreyra (INIMEC-CONICET), Córdoba, Argentina; and
Departamento de Química Biológica-CIQUIBIC, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba-CONICET, Argentina
Received January 28, 2000; accepted April 5, 2000
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ABSTRACT |
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Key Words: 2,4-D; neurotoxicity; developing neurons; microtubules; gangliosides; Golgi apparatus.
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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, 1980). 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., 1992). 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, 1980). 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, 1978
), 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, 1987) and processed for immunofluorescence as previously described (Cáceres et al., 1992
; DiTella et al., 1994
). The primary antibodies used were: a monoclonal antibody (mAb) against tyrosinated
-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
-tubulin (Cáceres et al., 1992
) 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., 1992
; DiTella et al., 1994
; Feiguin et al., 1994
; Paglini et al., 1998
).
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., 1991). 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., 1994
).
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., 1975). 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.
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RESULTS |
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DISCUSSION |
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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., 1994; Karecla and Kreis, 1992
; Kreis, 1990
; Scheel et al., 1990
) 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, 1990
).
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., 1999; Maccioni et al., 1999
). 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., 1990
) and on the biosynthesis of complex gangliosides (Ogiso et al., 1992
). 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., 1999
), 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.
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ACKNOWLEDGMENTS |
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NOTES |
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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)-4334074. E-mail: squiroga{at}dqbfcq.uncor.edu.
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