Department of Environmental Toxicology, One Shields Avenue, University of California, Davis, California 95616
Received August 9, 2000; accepted September 29, 2000
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ABSTRACT |
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Key Words: carbendazim; GTP binding; tubulin; microtubule assembly; microtubule-associated proteins; nocodazole; colchicine.
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INTRODUCTION |
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Until recently it was unclear whether BNL itself or its metabolite, CBZ, was responsible for the observed testicular toxicity. Lim and Miller (1997) measured testicular levels of BNL and CBZ and found that the extent of testicular damage could be directly correlated with the concentration of CBZ and not BNL. CBZ was also 2-fold more potent than BNL as a testicular toxicant. The testicular lesion following CBZ treatment is characterized by premature sloughing of spermatids, loss of Sertoli cell apical cytoplasm (Lim and Miller, 1997; Nakai and Hess, 1997
), and collapse of intermediate filaments (Hess and Nakai, 2000
).
The formation of MTs is a multi-step, temperature-dependent process. In the absence of a microtubule-organizing center (MTOC) such as the centrosome, the process starts with a nucleation event to generate the structures from which the MTs grow. Next is an elongation phase during which heterodimers of -ß tubulin assemble into polymers. Whether the polymers form MTs, sheets, ribbons, or other structures is influenced by the assembly conditions including buffer composition and the presence of microtubule-associated proteins (MAPs). Elevated temperatures (3037°C) stimulate assembly while lower temperatures cause MTs to disassemble. Magnesium ions and guanosine triphosphate (GTP) are generally required for MT assembly, with the latter bound at the exchangeable (E) and non-exchangeable (N) sites of the ß and
subunits, respectively. GTP is hydrolyzed during the formation of MTs but GTP hydrolysis per se appears not to be required for elongation of the polymer, which can be supported by non-hydrolyzable analogs of GTP (Dye and Williams, 1996
; O'Brien and Erickson, 1989
) as well as guanosine diphosphate (GDP; Carlier and Pantaloni, 1978). There appears to be a requirement for GTP in the initial formation of the nuclei from which the polymer grows, as this nucleation step is supported by tubulin with GTP-bound (GTP-tub) but not by GDP-tub (Karr et al., 1979
; O'Brien and Erickson, 1989
). Thus, an early step in the formation of MTs is the binding of GTP by tubulin, although the role of GTP hydrolysis in MT polymerization is complex and not fully understood (Dye and Williams, 1996
).
The present studies have examined the ability of CBZ to alter the assembly of MTs from tubulin prepared from rat brain and testis. This work evaluated the effects of MAPs on MT assembly in the presence and absence of CBZ, as well as the effects of CBZ on GTP binding. The effects of CBZ were compared with the response to the known MT disrupters, colchicine (CLC) and nocodazole (NOC).
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EXPERIMENTAL PROCEDURES |
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Tubulin preparation.
MAP-containing and MAP-free tubulin were prepared from rat brain and testis by 2 different methods, using either glycerol or sodium glutamate. To prepare tubulin-containing MAPs, the procedure of Shelanski et al. (1973) was used with modification. Rats were killed by CO2 inhalation and the brains and testes homogenized in MES buffer (0.1 M MES, pH 6.4, 1 mM EGTA, 1 mM MgCl2, 1 mM dithioerythritol, 1% aprotinin and 200 µM PMSF [phenylmethyl sulonylfluoride]). To enhance MT depolymerization, the homogenates sat on ice for 15 min prior to centrifugation at 100,000 x g for 60 min at 4°C. Tubulin was prepared by either one or two cycles of heat-induced assembly (37°C, 60 min) in the presence of glycerol (4 M) and GTP (1 mM). Each cycle was followed by centrifugation at 37°C at 100,000 x g for 30 min to pellet assembled MTs. The pellets were resuspended by sonication and incubated on ice to promote disassembly. Centrifugation (100,000 x g, 30 min at 4°C) removed protein aggregates. The tubulin-containing supernatant from the cold centrifugation step of the second cycle was filtered through glass wool, snap frozen in methanol/dry ice, and stored at 80°C.
For the preparation of tubulin substantially free of MAPs, 1 M glutamate was used in a batch preparation technique modified from Hamel and Lin (1981). The tissues were homogenized in 0.1 M MES (pH 6.75) with 1 mM EGTA, 0.5 mM MgCl2, 1% aprotinin and 200 µM PMSF. Samples sat on ice for 15 min prior to centrifugation at 100,000 x g as described above. GTP (0.1 M) was added to the supernatants, which were gently mixed for 60 min in the cold with DEAE-Sephacel (1 ml/10 g tissue) previously equilibrated with 1 M Na glutamate, pH 6.6. After the Sephacel had settled for 15 min on ice, the supernatant was removed and the resin was rinsed with washing buffer (1 M Na glutamate, 0.1 M NaCl, 0.1 mM GTP, pH 6.6) and pelleted at 100 x g. This wash step was repeated 2 times. Tubulin was eluted from the resin with elevated salt by incubation with 1 ml elution buffer (1 M Na glutamate, 1 M NaCl, 0.1 mM GTP, pH 6.6) on ice for 15 min with occasional gentle mixing. The resin was pelleted as before and the supernatant saved. This step was repeated for a total of 5 times. Glycerol (4 M) and GTP (1 mM) were added to the pooled supernatants and the tubulin allowed to polymerize at 37°C for 30 min. Assembled MTs were pelleted at 100,000 x g, 30 min 37°C. The pellets were resuspended in 1 M Na glutamate, sonicated, and allowed to depolymerize on ice for 30 min. Following a final centrifugation at 100,000 x g, depolymerized tubulin was collected in the supernatant and snap frozen. Residual nucleotides in the supernatant were removed by passage over Sephadex G25 minicolumns prior to use.
Preparation of MAPs.
MAPs were isolated according to Neely and Boekelheide (1988) as modified from Vallee (1982), snap frozen, and stored at 80°C. Briefly, MTs and associated MAPs were polymerized in the presence of taxol, glycerol and GTP, then pelleted by centrifugation through sucrose. MAPs were dissociated from the MTs with 750-mM NaCl and collected in the supernatant after pelleting the MTs. Prior to use, MAPs solutions were desalted by passage through Sephadex G25.
Assembly assay.
The assembly of tubulin into MTs was monitored turbidometrically at 350 nm in a Shimadzu recording spectrophotometer with a thermostatically regulated cuvette holder. Tubulin assembly mixtures included 2 mM Mg2+, and inhibitors or vehicles. No difference in the assembly was observed irrespective of whether MgCl2 or MgSO4 was used. Protein concentrations were determined with a bicinchoninic acid protein assay kit (Sigma, St Louis, MO) with BSA as standard. The reactions were initiated by the addition of 0.51.0 mM GTP and elevation of the cuvette temperature to 37°C. Optical densities were recorded at 2-min intervals through one or more cycles of heating to 37°C and cooling to less than 10°C. Tubulin samples were either preincubated or co-incubated with CBZ dissolved in dimethylformamide (DMF), NOC in dimethyl sulfoxide (DMSO), or CLC in water. The volume of added vehicle was 1% or less of the final reaction volume.
For experiments in which MAPs were added to MAP-free tubulin, MAPs:tubulin ratios from 1:4 to 1:15 (µg/µg) were tested with qualitatively similar results. The higher MAPs:tubulin ratios were used routinely as they gave more pronounced results.
[3H]-GTP binding to tubulin.
The effects of CBZ on the binding of GTP by tubulin were analyzed by the DEAE cellulose filter method of Borisy (1972). Tubulin was incubated with CBZ for 20 min on ice, prior to the addition of 5 µM [3H]-GTP. The reaction mixtures were diluted with PEM (80 mM PIPES, pH 6.8, 1 mM MgCl2, 1 mM ethylene glycol bis(aminoethyl ether) [EGTA]) and poured on to stacks of 3 DEAE cellulose filter disks (Whatman DE81, Fisher Scientific) on a vacuum manifold. The filters were rinsed twice with 4 ml 8 mM PEM containing 10 mM KCl to reduce nonspecific nucleotide binding to the filters. Protein-bound nucleotide on the filters was quantified by liquid scintillation spectroscopy after elution overnight in scintillation fluid (Emulsifier-Safe, Packard Inst., Meriden, CT).
Analysis of GTP- and GDP-tubulin.
The effects of CBZ on GTP hydrolysis were assayed by HPLC separation and quantitation of GDP and GTP according to Melki et al. (1996). MAP-free tubulin was incubated with 1 mM GTP for 30 min on ice. Unbound GTP was removed by passage over Sephadex G25 to yield a 1:1 GTP:tubulin complex. CBZ or vehicle (DMF) was added to the GTP:tubulin. After incubation for 240 min, 100 µl samples were transferred to 12 µl of ice cold 40% perchloric acid to precipitate protein, which was removed by centrifugation The ratio of GDP to GTP in solution was determined by HPLC on a Waters HPLC system equipped with an Alltech Adsorbosphere Nucleotide column. The samples were resolved by isocratic elution of the column with a mobile phase of methanol/60 mM NH4NHPO4 (25:75) containing 5 mM tetrabutylammonium sulfate as an ion-pair reagent.
Unless otherwise indicated, experiments were performed 3 times. Statistical significance of treatment effects was determined with a Student's 2-tailed t-test, p < 0.05.
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RESULTS |
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CBZ Acts on Tubulin
To determine whether CBZ's effects were primarily on assembling tubulin or whether it could disrupt already-formed MTs, CBZ (100 µM) was added to MAP-free tubulin in which MT assembly had reached a steady state at 37°C as shown by no further increase in the optical density. The cuvettes were cooled to <10°C, to allow disassembly, then another thermal cycle was initiated. Figure 6 depicts the changes in the optical density of tubulin preparations treated in this fashion. CBZ inhibited assembly when present at the start of the assembly reaction (Fig. 5A
), but had no apparent effect on assembled MTs (Fig. 6B
). However, once the MTs had disassembled, CBZ inhibited assembly during the second thermal cycle, suggesting that CBZ affected the initiation of assembly rather than directly disrupting MTs. This contrasts with the effects of NOC on MTs. When NOC (12.5 µM) was added to MTs at steady state there was an immediate loss of turbidity, indicating depolymerization of the MTs (Fig. 6A
). No MT assembly was seen during the second thermal cycle in the presence of NOC. Similar to CBZ, CLC (40 µM) did not cause depolymerization of the MTs and did inhibit MT assembly during the second cycle. The cold-induced depolymerization observed after CLC was not as extensive as that observed after either CBZ or NOC.
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DISCUSSION |
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The selection of CLC and NOC as positive controls in the present study was based in part on published competitive binding studies with CLC. From these studies it had been suggested that the benzimidazoles bind in or near the CLC binding site and perhaps operate through a similar mechanism (Davidse and Flach, 1977; Russell et al., 1992
). However, several observations from the present work suggest dissimilar mechanisms of action between the two compounds, CBZ and NOC. When added to preformed MTs, only NOC stimulated their depolymerization whereas CBZ and CLC had no apparent effect, suggesting a differential ability of MTs to bind NOC versus CBZ and CLC. Indeed, the fact that CLC binds free tubulin rather than MTs has been used to explain the observed stability of MTs exposed in vitro to high concentrations of CLC (Farrell and Wilson, 1984
). CBZ also appears to bind only free tubulin, since it was not until the MTs disassembled that CBZ and CLC were able to prevent further re-polymerization under assembly-promoting conditions. NOC and CBZ are also dissimilar in that NOC is reportedly much more tightly bound by tubulin (Friedman and Platzer, 1978
). Furthermore, whereas NOC has been associated with an increase in GTPase activity (Mejillano et al., 1996
), there was no evidence that CBZ stimulated GTP hydrolysis in the present studies.
In contrast to NOC, CBZ and CLC were more alike, since neither caused depolymerization of MTs and both inhibited subsequent repolymerization. In addition, the in vivo effects of CBZ and CLC are similar in that CLC can induce pathological changes in the testis (Allard et al., 1993) that resemble the premature sloughing of germ cells seen with CBZ (Hess et al., 1991
; Nakai et al., 1993
). There are, however, differences between CBZ and CLC associated with stage-specific sensitivity. Allard et al. (1993) reported that stages IXXIV seminiferous tubules were the most sensitive to CLC. With CBZ, sloughing was seen starting in stage VII (Parvinen and Kormano, 1974
), one h after exposure, and from stage VI onward, 2 h post-exposure (Nakai et al., 2000
). At 3-h post-CBZ exposure, sloughing was seen in all stages except IIIV (Nakai and Hess, 1994
). In the testis, the stage specific sensitivity of Sertoli cells to CBZ vs. CLC may be related to the stage-dependent presence of various MAPs and/or chaperone proteins. Several of the MAPs are known to stabilize MTs or promote assembly upon binding near the carboxyl terminus of tubulin (Maccioni and Cambiazo, 1995
). The modulation of CBZ's assembly inhibition by these MAPs would therefore likely be indirect and involve allosteric effects. On the other hand, certain of the chaperonins, recently recognized for their involvement in the assembly of MTs as well as the folding and heterodimerization of the tubulins, have subunits that bind to and co-purify with tubulin (Roobol et al., 1999
). It remains to be determined which of these MAPs or MAP-like proteins modulate CBZ binding and the mechanisms involved.
It was noted that, when added to assembling MAP-free tubulin shortly (2 min) after the addition of GTP, CBZ was a much less potent inhibitor than when it was present prior to GTP addition. The dependence of inhibition on the sequence of the addition of CBZ and GTP indicates that CBZ does not displace bound GTP. Rather it appears that CBZ prevents GTP binding, thereby interfering with the initiation of assembly. This scenario is further supported by the competitive binding experiments in which CBZ inhibited GTP binding to tubulin in vitro in a dose-dependent fashion. The IC50 of 74 µM that we obtained for CBZ inhibition of GTP binding in the brain is in good agreement with the IC50 of 90 µM reported by Friedman and Platzer (1978) for the inhibition of brain tubulin assembly by CBZ. Similarly, if CBZ inhibits GTP binding by direct competition for a common or overlapping binding site, then excess GTP should competitively prevent CBZ inhibition. Indeed, in assembly assays in which excess GTP was added to MTs after the addition of CBZ, the inhibition of assembly during the second cycle was significantly reduced.
A mechanism for binding competition between CBZ and GTP is provided by the observation that the amino acids involved in CBZ binding in fungal tubulin overlap the GTP binding site. Based on studies of fungal sensitivity and resistance to CBZ following site-directed mutagenesis, amino acids 6, 165167, and 198200 are thought to be involved in the binding of CBZ (Fujimura et al., 1992; Jung et al., 1992
). Comparison of published tubulin sequence data indicates that these amino acids are conserved between fungal and mammalian tubulins. According to the model presented by Nogales et al. (1998), amino acids 38 are thought to contact phosphates on GTP/GDP while amino acids 166171 are very near the ribose. This is in concordance with the photolabeling of tubulin within amino acids 335 by 8-azido-GTP (Jayaram and Haley, 1994
) and amino acids 155174 by GTP (Hesse et al., 1987
). Thus GTP and CBZ binding sites overlap at amino acid 6 and in the region of amino acids 165167.
The levels of CBZ in these experiments that inhibit assembly and GTP binding were approximately 5-fold lower than those measured in testis following in vivo ip exposure to CBZ, which caused testicular lesions in rats (Lim and Miller, 1997). This further enhances the significance of GTP-binding inhibition in the etiology of CBZ toxicity. In these experiments, the responses of brain and testis to CBZ inhibition of tubulin assembly and GTP binding in vitro were similar. However, this does not rule out a differential tissue-specific sensitivity to CBZ in vivo. For example, it is well known that in the various stages of spermatogenesis there is differential expression of proteins. This would also offer an explanation of the stage-specific toxicity reported for CBZ treatment. In neuronal tissue, there is no corollary to the cyclical expression of MAPs in the testis.
In summary, we have found that CBZ is an effective inhibitor of the in vitro assembly of brain and testis tubulin, an effect that is strongly moderated by MAPs. Our data support a mechanism for CBZ action in which CBZ inhibits the binding of GTP by tubulin, thereby interfering with the initiation of tubulin polymerization and the formation of MTs.
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NOTES |
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