* Department of Environmental Toxicology, University of California, One Shields Avenue, Davis, California 95616;
Animal Sciences Laboratory, University of Illinois, 1207 W. Gregory Drive, Urbana, Illinois 61801; and
Department of Veterinary Biosciences, University of Illinois, 2001 S. Lincoln, Urbana, Illinois 61802
Received February 15, 2002; accepted April 30, 2002
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ABSTRACT |
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Key Words: benzimidazoles; carbendazim; colchicine; microtubules and tubulin; microtubule disrupting agents; microtubule associated proteins; Sertoli cells.
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INTRODUCTION |
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Carbendazim has been demonstrated to be the active toxicant responsible for the observed testicular toxicity. In a previous study, testicular levels of carbendazim, and not benomyl, were directly correlated with the extent of testicular damage in rats (Lim and Miller, 1997a). Carbendazim was also found to be a two-fold more potent testicular toxicant than was benomyl (Lim and Miller, 1997a
). Carbendazim is thought to act as a fungicide by binding to the colchicine binding site of fungal tubulin, resulting in inhibition of microtubule assembly in vitro (Davidse, 1986
; Davidse and Flach, 1977
). It has been proposed that carbendazim causes testicular toxicity in mammals by a similar mechanism that disrupts microtubules (Davidse and Flach, 1977
; Russell et al., 1992
). Recently, carbendazim was shown to disrupt microtubules in freshly isolated rat seminiferous tubules in situ, evidenced by an increase in soluble pools of tubulin (Correa and Miller, 2001
). This increase in tubulin subunit levels may link the characterized histological damage (Hess and Nakai, 2000
) with the ability of carbendazim to disrupt microtubule assembly in vitro (Davidse and Flach, 1977
; Friedman and Platzer, 1978
; Ireland et al., 1979
; Russell et al., 1992
; Winder et al., 2001
).
Mammalian species exhibit differential sensitivity to the benzimidazoles (Davidse, 1986). In a multigenerational study, chronic treatment of rats and hamsters with carbendazim caused reproductive damage to rats based on several endpoint measurements, yet in hamsters, only sperm measures were affected (Gray et al., 1990
). In another study that investigated sperm morphology, mouse sperm appeared to be less sensitive to the effects of carbendazim than rat sperm (Evenson et al., 1987
). However, early time points and single dose effects of carbendazim were not evaluated in these studies (Evenson et al., 1987
; Gray et al., 1990
). It is interesting that prepubertal rats have been determined to be less sensitive to the effects of carbendazim compared to adult rats (Carter et al., 1984
; Lim and Miller, 1997b
). This appears to be due, at least in part, to a relatively low level of detectable carbendazim in the prepubertal testis compared to the adult rat testis (Lim and Miller, 1997b
). Clearly, species differences in carbendazim distribution, metabolism, and elimination have to be considered when evaluating species-specific toxicity.
Colchicine, a well described microtubule disruptor, also causes testicular damage in rats, including sloughing, similar to that caused by carbendazim (Allard et al., 1993; Russell et al., 1981
). However, the degree to which colchicine causes reproductive damage in the mouse is not well characterized. In one study, injection of colchicine into mouse testes each day for up to 9 days caused sperm abnormalities and disruption of microtubules in the seminiferous epithelium (Handel, 1979
).
In the current study, we have investigated the sensitivity of the mouse testis to carbendazim and colchicine, and potential mechanisms underlying any differential effects of these agents compared to the rat. The effects of these two microtubule disruptors on seminiferous tubule structure were characterized and carbendazim levels in the testis were determined. The possibilities that a reduced level of carbendazim reaches the mouse testis or that the mouse cytoskeleton is relatively insensitive to the effects of carbendazim were addressed.
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MATERIALS AND METHODS |
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Chemicals.
HPLC grade water and methanol were purchased from Fisher Scientific (Pittsburgh, PA). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise indicated.
Administration of chemicals.
For histopathological assessment, carbendazim was injected into mice (ip) at 2000 mg/kg carbendazim suspended in corn oil, and controls were injected with corn oil. The animals were asphyxiated by CO2 inhalation 2, 3, or 6 h after treatment, and tissues were fixed and processed for microscopy as described below. For determination of testicular levels of carbendazim, 2000 mg/kg carbendazim suspended in corn oil was injected ip and testes were collected 5, 15, 30, 60, 90, and 120 min following injection. For comparison, 164 mg/kg carbendazim suspended in corn oil was injected ip into rats and testes were collected 15 min after injection. Tissues were frozen immediately in liquid nitrogen and stored at 80°C prior to HPLC analysis.
Colchicine was injected intratesticularly into mice at 5.3 or 117.6 µg colchicine/g testis (dissolved in 25 µl PBS), and into rats at 5.6 µg colchicine/g testis (dissolved in 50 µl PBS). The contralateral testis on each animal was injected with a 25 µl (mice) or 50 µl (rat) volume of PBS and served as controls. Animals were sacrificed 6 h after treatment and testes were processed for histology or immunohistochemistry.
Histology.
Testes collected after carbendazim and colchicine treatment were immersion fixed in 10% phosphate-buffered formalin and tissue blocks were embedded with glycol methacrylate and sectioned (2.5 µm). Sections were stained by the periodic acid-Schiff reaction and hematoxylin (PAS-H) and viewed by routine light microscopy. Histopathological damage was assessed by examination of 100 seminiferous tubule cross sections per testis. Sections were evaluated for the following endpoints: vacuolization (appearance of vacuoles in the seminiferous epithelium), and sloughing (loss of the apical portions of Sertoli cells and attached germ cells). The percentage of normal tubules and of tubules with the described histological endpoints was determined.
Immunohistochemistry.
Testes collected after carbendazim and colchicine treatment were immersion fixed in 1 or 4% paraformaldehyde and tissue blocks were processed for paraffin sectioning. Sections were incubated in 0.3% hydrogen peroxide in methanol for 15 min to block endogenous peroxidase activity, followed by a 10-min incubation in 10% normal goat serum to prevent nonspecific binding of antibodies. Sections were incubated with either anti-tyrosinated tubulin monoclonal antibody (diluted 1:1000, TUB-1A2, Sigma) or anti-ß tubulin monoclonal antibody (diluted 1:100, Chemicon International, Temecula, CA) for 2 h at room temperature or overnight at 4°C. Control sections were incubated as above without primary antibody. All sections were incubated with biotinylated goat anti-mouse IgG secondary antibody (diluted 1:100, DAKO, Carpinteria, CA) followed by incubation with avidin-biotinylated peroxidase complex (Vectastain ABC kit, Vector laboratories, Burlingame, CA). Positive reactions were visualized with diaminobenzidine and hydrogen peroxide.
Carbendazim levels in the testis.
Mice testes were collected at 5, 15, 30, 60, 90, and 120 min postinjection, and rat testes were collected 15 min postinjection, as described above. Testes from each animal were weighed and homogenized in two volumes of 50 mM potassium phosphate buffer, pH 7.4, using a glass homogenizer and a teflon pestle. Homogenates were mixed with two volumes of methanol, vortexed for 1 min, and centrifuged at 2000 x g for 10 min at 4°C. The supernatants were removed and 100 µl aliquots were analyzed by HPLC based on the method of Lim and Miller (1997b). Carbendazim was separated by reverse-phase HPLC on a C18 column using a gradient solvent system. The flow rate was 1.0 ml/min, and detection was by UV absorption at 280 nm. Initial conditions in the gradient solvent system were 70% water: 30% methanol, followed by a linear gradient to 30% water:70% methanol over 5 min. This ratio of solvents was maintained for 5 min, followed by a linear gradient back to 70%:30% over 5 min and subsequent equilibration for an additional 15 min. The retention time for carbendazim was 13.7 min.
Standards ranging in concentration from 50 to 1600 ng carbendazim in 100 µl were made up in the same buffer:methanol ratio as testis samples, and used to construct standard curves of concentration versus area under the curve. The nmoles of carbendazim in testis samples were calculated from standard curves.
Statistics.
Statistical significance of treatment effects was determined with a Students two tailed t-test; p values < 0.05 were considered to be statistically significant.
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RESULTS |
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Effects of Carbendazim and Colchicine on Sertoli Cell Microtubules
To determine if mouse Sertoli cell microtubules are sensitive to carbendazim, mouse testis sections were stained with either tyrosinated tubulin or ß tubulin antibodies. In Figure 3
, testis sections obtained 2 h after injection of carbendazim (2000 mg/kg) or control are shown. In the control (Fig. 3A
),
tubulin occurs in tubule cross-sections in a spoke-like pattern characteristic of the prominent microtubules of the Sertoli cell cytoskeleton. Virtually identical staining patterns of tyrosinated
tubulin are apparent in seminiferous tubule sections from a mouse testis that was treated with carbendazim (Fig. 3B
). The "spokes" observed reflect microtubules extending from the basal to the apical portion of Sertoli cells, perpendicular to the basement membrane of each seminiferous tubule. Similarly, the characteristic pattern of
tubulin staining was observed in tubule sections prepared from mice 6 h after injection of carbendazim (data not shown). Figure 4
contains testis sections obtained 2 h after injection of carbendazim that were stained for ß tubulin. Control (Fig. 4A
) and treated (Fig. 4B
) testes are virtually identical and reveal the pattern of tubulin staining characteristic of the extensive microtubule network of Sertoli cells. Sections prepared from testes 6 h after injection of carbendazim also displayed ß tubulin staining indistinguishable from controls (data not shown). The slight differences in staining between adjacent seminiferous tubule sections in Figures 3 and 4
reflect the various stages that were bisected in the testis sections pictured. The immunohistochemical results are in agreement with the histological data, thus supporting the hypothesis that carbendazim does not induce sloughing or loss of microtubule structure in the mouse testis.
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DISCUSSION |
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In the rat, a single injection of 164 mg/kg carbendazim is capable of eliciting testicular toxicity, with evidence of detachment and sloughing of germ cells beginning as early as 1 h after treatment (Lim and Miller, 1997a) and continuing for several hours. In the present study, the seminiferous epithelium was undamaged and identical to controls at 3 h and 6 h after injection of carbendazim to mice. The extensive microtubule network characteristic of Sertoli cells was also intact based on immunohistochemical staining of tyrosinated
and ß tubulin, in agreement with the histological results. In contrast, it has been reported that the rat Sertoli cell microtubule cytoskeleton is severely disrupted after carbendazim treatment, evidenced by loss of tyrosinated
tubulin staining (Hess and Nakai, 2000
). The reason why the rat testis is sensitive to carbendazim while the mouse is unaffected even at very high doses is unknown.
There are at least two possible hypotheses to explain why the mouse seminiferous epithelium is insensitive to the microtubule disruptor carbendazim: either carbendazim does not enter the testis (or does so at very reduced amounts), or it does enter the testis and the mouse possesses a mechanism to prevent toxicity. To investigate these possibilities, the level of carbendazim in whole mouse testis homogenates was determined over a 2-h period using HPLC. The maximal concentration of carbendazim measured in the testis, 375 nmol/g testis, occurred at approximately 5 min postinjection with the level decreasing over time as carbendazim was metabolized. This value is comparable to a previously determined maximum value for carbendazim (280 nmol/g testis) in the rat testis, measured 15 min postinjection (Lim and Miller, 1997a). The result of this difference is a relatively longer exposure time in the rat testis to high levels of carbendazim, which might conceivably allow more testicular damage to occur. However, the detectable level of carbendazim in mouse testis at 2 h of exposure was approximately twice as high as previously determined for the rat (Lim and Miller, 1997a
). Whether these differences in carbendazim absorption and metabolism contribute to differential sensitivities to this microtubule-disrupting agent will require further investigation. Nevertheless, it is apparent that carbendazim is present in the mouse testis at concentrations greater than or equal to that which was present in the rat testis, yet it does not cause testicular damage.
In contrast to the lack of effect of carbendazim, colchicine did cause damage to the mouse seminiferous epithelium, including germ cell sloughing and loss of Sertoli cell microtubules based on the loss of thick bundles or "spokes" of tyrosinated tubulin and ß tubulin. However, the amount of colchicine necessary to elicit testicular damage was approximately 20 times higher than previously described for the rat. When a dose of colchicine that causes extensive damage in rat testis was administered to mice, testis sections appeared normal except that seminiferous tubule lumens were larger than in controls (Fig. 2B
). Since colchicine was injected directly into mice testes, the question of whether it is present in the testis or not is ruled out. Thus, there must be another mechanism at play that contributes to the reduced sensitivity of the mouse testis toward colchicine. For example, the high sensitivity of the rat to the effect of colchicine could be the result of greater binding affinity at the colchicine binding site of tubulin. Moreover, the same could be true for carbendazim since it also interacts at the colchicine binding site. Altogether, the data demonstrate that the mouse seminiferous epithelium is less sensitive than the rat to microtubule disruption by either colchicine or carbendazim.
Overall, our present study has demonstrated that the testicular toxicity of two microtubule disruptors, carbendazim and colchicine, can be very different between species. Moreover, it was previously shown that the effects of carbendazim in the rat are specific for the testes (Sherman et al., 1975) and, within the testis, damage is found only during specific stages of spermatogenesis (Hess and Nakai, 2000
). Early studies demonstrated that some benzimidazoles inhibit assembly of brain microtubules in vitro, although with much less sensitivity compared to colchicine and other known microtubule-disrupting agents (Friedman and Platzer, 1978
; Ireland et al., 1979
). It is possible that the mechanisms operating to protect the brain and particular stages of spermatogenesis could be protecting the mouse testis from carbendazim-induced disruption of microtubules. Previous work from this laboratory has shown that addition of microtubule associated proteins (MAPs) to in vitro preparations of rat MAP-free tubulin abolished the inhibitory effect of carbendazim on microtubule assembly (Winder et al., 2001
). Therefore, it is reasonable to propose that the sensitive stages of spermatogenesis in the rat could have a deficiency of protective MAPs, i.e., one or more MAPs that block the ability of carbendazim to bind tubulin. Perhaps the brain and mouse testis maintain a protective profile of MAPs and thus are insensitive to microtubule disruption after carbendazim treatment. These and other possibilities need further investigation in order to more clearly define the mechanisms underlying the species-specific and tissue-specific sensitivity toward the microtubule disrupting agents, carbendazim and colchicine.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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