Microtubule assembly is altered following covalent modification by the n-hexane metabolite 2,5-hexanedione.
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Anilino Naphthalenesulfonates↗
Biomedical subjects
K Boekelheide
Publications and source records attributed to K Boekelheide.
2,5-Hexanedione exposure alters the rat Sertoli cell cytoskeleton. I. Microtubules and seminiferous tubule fluid secretion.
2,5-Hexanedione (2,5-HD) is a testicular and nervous system toxicant with an unknown mechanism of action. In this study, the effects of 2,5-HD on seminiferous tubule fluid (STF) secretion, testis morphology, and tubulin distribution were examined. Charles River CD rats (200 g) were exposed to 1% 2,5-HD in the drinking water for 5 weeks followed by a 3-week recovery period. STF secretion was measured by efferent duct ligation, and testis cross sections were prepared at 2, 3, 3.43, 3.57, 3.86, 4, and 8 weeks after beginning exposure. A dramatic inhibition of STF secretion was observed between Weeks 3 and 4. The inhibition of STF secretion occurred following simultaneous changes in Sertoli cell and elongate spermatid morphology but prior to changes in round spermatid morphology. Also, alterations in seminiferous tubule tubulin distribution were observed with kinetics similar to those for changes in seminiferous tubule morphology. This temporal sequence suggests a model of 2,5-HD-induced injury in which populations of germ cells are differentially sensitive to impairment of Sertoli cell function.
Animals↗
2,5-Hexanedione exposure alters the rat Sertoli cell cytoskeleton. II. Intermediate filaments and actin.
The effects of 2,5-hexanedione (2,5-HD) exposure on the expression of intermediate filament and actin proteins in adult rat testis was examined during progressive stages of testicular injury. The distribution of vimentin within testis cross sections was examined using immunohistochemistry while rhodamine-labeled phalloidin was used to localize filamentous actin. Keratin was not detected in immunoblots of intermediate filament proteins tested with an antikeratin antibody to investigate the possible reexpression of the prepubertal intermediate filament protein keratin in 2,5-HD-exposed adult testes. However, 2,5-HD exposure did result in a progressively abnormal distribution of actin and vimentin within the seminiferous epithelium with the appearance of a high-molecular-weight protein which was vimentin immunoreactive and not present in control testes.
Actins↗
2,5-Hexanedione-treated tubulin microinjected into sea urchin zygotes induces mitotic abnormalities.
Zygotes of Lytechinus pictus and Lytechinus variegatus were microinjected with 2,5-hexanedione (2,5-HD)-treated tubulin prior to the first mitotic cycle. Mitotic spindles were small with a well-defined metaphase plate, but poor birefringence and poor astral development. Abnormalities were observed in chromosome movement at anaphase and cytokinesis. Neither microinjections of untreated tubulin or 3-acetyl-2,5-hexanedione-treated tubulin, nor incubation of zygotes in 2,5-HD-containing sea water produced abnormalities. The results can be explained in terms of the nondissociating properties of 2,5-HD-treated tubulin. 2,5-HD-treated tubulin dissociates slowly from microtubules, a property which, besides favoring the formation of stable microtubules, allows this tubulin to induce microtubule assembly when present in substoichiometric amounts. These characteristics have been implicated as a cause of 2,5-HD-induced Sertoli cell dysfunction. The effect of 2,5-HD-treated tubulin on microtubule dynamics in sea urchin zygotes may bear similarities to the effects of 2,5-HD treatment in vivo on Sertoli cell microtubules.
Animals↗
HMW-2, the Sertoli cell cytoplasmic dynein from rat testis, is a dimer composed of nearly identical subunits.
The ultrastructure and biochemical characteristics of HMW-2, the Sertoli cell cytoplasmic dynein isolated from rat testes, were analyzed. Electron microscopic studies revealed a two-headed two-stem structure with dimensions very similar to other dyneins. We found that, like other cytoplasmic dyneins, both heads have an approximately spherical shape with a central cavity. Heavy chain analysis suggested the presence of only one type of heavy chain, a finding that was supported by the simple Michaelis-Menten kinetics displayed by the HMW-2-associated ATPase activity. In addition, dissociation of the HMW-2 complex resulted in a single type of dynein subunit sedimenting at 11.8 S. This fraction contained all the polypeptides present in the undissociated HMW-2. Ultrastructurally the HMW-2 subunits were composed of one globular domain with a tail. The simplest interpretation is that HMW-2 is a dimer of nearly identical subunits, each containing one heavy chain, one 90-kDa intermediate chain, and two light chains.
Adenosine Triphosphatases↗
Selection of a nucleation-promoting element following chemical modification of tubulin.
Following a 16-h incubation with a large excess of 2,5-hexanedione (2,5-HD) while in the assembled state, bovine brain tubulin contained a powerful nucleating component, the presence of which lowered the dissociation rate from 83 s-1 for untreated tubulin to 13 s-1 for 2,5-HD-treated tubulin. This nucleating component could be selectively concentrated by sequential stringent (conditions of low temperature and low tubulin concentration) cycles of assembly and disassembly. In 2-(N-morpholino)ethanesulfonic acid buffer without glycerol, the critical concentration of assembly of untreated tubulin (2.4 mg/mL) was 19 times higher than that of 2,5-HD-treated tubulin subjected to three sequential stringent cycles of assembly and disassembly (0.13 mg/mL). This highly nucleating 2,5-HD-treated tubulin preparation could both copolymerize with untreated tubulin and seed subcritical concentration assembly of untreated tubulin. Experiments to define the assembly-altering component have identified structural alterations to the alpha-tubulin monomer. While the alpha-tubulin subunit of native untreated tubulin dimer contained no chymotryptic cleavage sites, the native 2,5-HD-treated alpha-tubulin subunit was cleaved by chymotrypsin to yield a 37-kDa C-terminal fragment.
Animals↗
The Sertoli cell cytoskeleton: a target for toxicant-induced germ cell loss.
Numerous studies in recent years have elucidated fundamental properties of axoplasmic structure, biochemistry, and function. The structural role of the cytoskeletal elements, the orientation of MTs within the axon, the phenomenon of MT-dependent transport, and the identity and direction of movement of two MT motors--kinesin and MAP-1C--have been revealed. For many years to come, researchers investigating the structure and function of the Sertoli cell cytoskeleton will be able to adapt techniques gleaned from work on the axonal cytoskeleton. Innovative thinking will be required to apply these techniques to the special circumstances of the male reproductive system; however, the underlying questions are similar. For example, knowledge of several fundamental properties of transport processes in the Sertoli cell would facilitate the toxicologic evaluation of this system. What is the orientation of MTs within the Sertoli cell cytoplasm? Are the fast-growing (+) ends of all MTs in the Sertoli cell cytoplasm directed toward the lumen? This is an important question because the direction of MT-dependent transport involving known MT motors is dependent upon the MT orientation. Which of the Sertoli cell transport pathways are MT-dependent pathways? What are the MT motors involved in these pathways? Ultrastructural examination following exposure to specific cytoskeleton-disrupting agents has highlighted the importance of AFs, IFs, and MTs in the Sertoli cell. Future research will focus on the nature of those molecules which integrate these cytoskeletal components into a dynamic whole, the regulatory systems which control this integration, and the role of an integrated cytoskeleton in Sertoli cell function and testicular homeostasis. Toxicology will be an active participant in this process of scientific discovery. The selective nervous system and testicular toxicants may be useful tools in revealing similarities in the cytoskeletal organization of these apparently disparate organ systems. By searching for common targets in the testis and nervous system, the mechanisms of action of these agents may be more easily, and more confidently, determined.
Animals↗
The rate of 2,5-hexanedione intoxication, not total dose, determines the extent of testicular injury and altered microtubule assembly in the rat.
Charles River CD rats (220 g) were intoxicated with 1.0, 0.5, or 0.25% 2,5-hexanedione (2,5-HD) in the drinking water for a total of 21, 35, or 69 days, respectively. All rats received a total dose of 131 +/- 2 mmol/kg 2,5-HD at dose rates ranging from 1.9 to 6.1 mmol/kg/day. Rats were sacrificed 4 weeks after ending intoxication to evaluate the extent of testicular injury. An exposure rate of 6.1 mmol 2,5-HD/kg/day produced uniformally low testis weights (49% of control) and severe germ cell depletion, while exposure at 1.9 mmol/kg/day gave normal testis weights and histology. Exposure at the intermediate dose rate of 3.8 mmol 2,5-HD/kg/day produced an intermediate degree of testicular injury. In a separate experiment, testis pyrrole content and microtubule assembly behavior were measured in rats exposed to 2,5-HD at the various dose rates for 3 weeks. The rate of intoxication determined the extent of biochemical abnormality. Rats exposed to 1.0, 0.5, or 0.25% 2,5-HD had microtubule nucleation times 55, 63, and 72% of control and pyrrole contents equivalent to 2.14, 1.40, and 1.18 nmol 2,5-dimethylpyrrole/mg testis protein. These data demonstrate that 2,5-HD-induced testicular injury, unlike the nervous system toxicity, is dependent upon the rate of intoxication independent of total dose.
Animals↗
Rat testis during 2,5-hexanedione intoxication and recovery. I. Dose response and the reversibility of germ cell loss.
The histopathology of the testicular injury induced by 2,5-hexanedione (2,5-HD) exposure was examined in the rat. Charles River CD rats (200 g) were intoxicated by consuming 1% 2,5-HD in the drinking water or by intraperitoneal injection of the toxicant. Both neurotoxic and subneurotoxic exposures were studied, the total dose ranging from 40 to 211 mmol/kg. The following results were obtained: (1) there was a time delay between administration of the toxicant and development of the testicular injury, (2) Sertoli cell vacuolation in stages associated with the meiotic metaphase was the first histological sign of cellular injury at all doses, (3) subneurotoxic doses produced selective defects in germ cells in stages I-VIII of the spermatogenic cycle, (4) both subneurotoxic and neurotoxic doses produced germ cell necrosis and generalized sloughing of germ cells, and (5) intensive intoxication followed by a 17-week recovery period resulted in an absence of all postspermatogonial germ cells from the seminiferous epithelium of three of five treated rats. These data demonstrate that 2,5-hexanedione-induced testicular atrophy occurs at exposure levels below those producing clinical neurotoxicity and that, within the time frame of this study, the testicular injury is at least partially irreversible.
Administration, Oral↗
Rat testis during 2,5-hexanedione intoxication and recovery. II. Dynamics of pyrrole reactivity, tubulin content, and microtubule assembly.
Charles River CD rats (200 g) were intoxicated with 1% 2,5-hexanedione (2,5-HD) in the drinking water for 5 weeks followed by a 17-week recovery period. Pyrrole reactivity of testis proteins increased early during intoxication and then returned toward normal during recovery. Testis tubulin content first increased as germ cells were lost and then fell over time while atrophy was maintained. Purified testis tubulin demonstrated a decreased nucleation time for microtubule assembly at 2 weeks, maintained this alteration throughout intoxication, and then returned to normal assembly kinetics during recovery. The assembly abnormality was accompanied by the presence of a unique crosslinked tubulin species. These findings support the hypothesis that alterations in Sertoli cell microtubules result in germ cell loss following 2,5-HD exposure.
Administration, Oral↗
Sertoli cell processes have axoplasmic features: an ordered microtubule distribution and an abundant high molecular weight microtubule-associated protein (cytoplasmic dynein).
Microtubules in the cytoplasm of rat Sertoli cell stage VI-VIII testicular seminiferous epithelium were studied morphometrically by electron microscopy. The Sertoli cell microtubules demonstrated axonal features, being largely parallel in orientation and predominantly spaced one to two microtubule diameters apart, suggesting the presence of microtubule-bound spacer molecules. Testis microtubule-associated proteins (MAPs) were isolated by a taxol, salt elution procedure. Testis MAPs promoted microtubule assembly, but to a lesser degree than brain MAPs. High molecular weight MAPs, similar in electrophoretic mobilities to brain MAP-1 and MAP-2, were prominent components of total testis MAPs, though no shared immunoreactivity was detected between testis and brain high molecular weight MAPs using both polyclonal and monoclonal antibodies. Unlike brain high molecular weight MAPs, testis high molecular weight MAPs were not heat stable. Testis MAP composition, studied on postnatal days 5, 10, 15, and 24 and in the adult, changed dramatically during ontogeny. However, the expression of the major testis high molecular weight MAP, called HMW-2, was constitutive and independent of the development of mature germ cells. The Sertoli cell origin of HMW-2 was confirmed by identifying this protein as the major MAP found in an enriched Sertoli cell preparation and in two rat models of testicular injury characterized by germ cell depletion. HMW-2 was selectively released from testis microtubules by ATP and co-purified by sucrose density gradient centrifugation with MAP-1C, a neuronal cytoplasmic dynein. The inhibition of the microtubule-activated ATPase activity of HMW-2 by vanadate and erythro-(2-hydroxy-3-nonyl)adenine and its proteolytic breakdown by vanadate-dependent UV photocleavage confirmed the dynein-like nature of HMW-2. As demonstrated by this study, the neuronal and Sertoli cell cytoskeletons share morphological, structural and functional properties.
Adenosine Triphosphatases↗
2,5-Hexanedione alters microtubule assembly. I. Testicular atrophy, not nervous system toxicity, correlates with enhanced tubulin polymerization.
Charles River CD rats (200 g) were divided into three groups receiving either 1% 2,5-hexanedione (2,5-HD) or 0.035% 3,4-dimethyl-2,5-hexanedione (DMHD) in the drinking water or water alone (control) for 4 weeks. The two treated groups experienced similar nervous system dysfunction and systemic toxicity. Testicular toxicity, as evidenced by histological changes and decreased testis weight, was present only in 2,5-HD-treated rats. Tubulin was purified from brain and testis and assembly properties were determined. Purified brain and testis tubulin derived from the 2,5-HD-intoxicated rats displayed altered assembly with a shortened nucleation phase and more rapid rate of elongation. Brain tubulin from DMHD-intoxicated rats displayed assembly behavior similar to controls, while testis tubulin from DMHD-intoxicated rats displayed assembly behavior intermediate between the control and 2,5-HD tubulin preparations. The presence of gamma-diketone-induced assembly alterations following in vivo intoxication was accompanied by the formation of a high-molecular-weight protein identified as crosslinked tubulin. From these data, we conclude that microtubule assembly alterations are not etiologic in the development of nervous system dysfunction following intoxication but may represent the biochemical mechanism of 2,5-HD-induced testicular atrophy.
Animals↗
2,5-Hexanedione alters microtubule assembly. II. Enhanced polymerization of crosslinked tubulin.
The toxic syndrome resulting from in vivo exposure to n-hexane or n-hexane derivatives may, in part, be a manifestation of altered tubulin and microtubule properties. The effect of in vitro gamma-diketone derivatization was first studied using purified bovine brain tubulin and the results were then verified in tubulins purified from target organs of an experimental species. Microtubule assembly and structure were modified after in vitro incubation with 2,5-hexanedione (2,5-HD) as follows: 2,5-HD derivatization of purified tubulin resulted in an alteration in microtubule assembly kinetics, most prominently a decrease in the length of the nucleation phase, the alteration in assembly kinetics was accompanied by the formation of a covalently crosslinked tubulin dimer, mixing experiments which combined different proportions of control and treated tubulin showed that only a small amount of derivatized tubulin need be present to induce altered assembly properties, and as a result of the more rapid nucleation phase, a greater number of nucleating seeds produced more numerous and shorter assembled polymers. In vitro incubation with the 2,5-HD congener 3,4-dimethyl-2,5-hexanedione produced similar alterations in microtubule assembly. Thus, both the kinetics of tubulin polymerization and the morphology of the final assembly product were modified by in vitro gamma-diketone incubation.
Animals↗
Microtubule assembly inhibition by porphyrins and related compounds.
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Colchicine↗
Covalent crosslinking of neurofilaments in the pathogenesis of n-hexane neuropathy.
These studies test the hypothesis that in n-hexane neuropathy the gamma-diketone metabolite 2,5-hexanedione (2,5-HD) results in covalent crosslinking of neurofilaments via nucleophilic attack on oxidized pyrrole rings formed from the reaction of 2,5-HD with epsilon-amino groups of lysyl residues. The 2,5-HD analogue and gamma-diketone,3,4-dimethyl-2,5-hexanedione (DMHD), was found to result in more rapid pyrrole formation, pyrrole autoxidation, and protein crosslinking when compared with 2,5-HD. DMHD was 20-30 times more potent than 2,5-HD in producing hindlimb paralysis. Following 2,5-HD intoxication the neurofilament filled axonal swellings were found in the distal, subterminal axon. After treatment with DMHD, swellings were present in the proximal axon, similar to those seen after intoxication with beta,beta'-iminodipropionitrile (IDPN). DMHD was proposed as a connecting link between the proximal neurofilamentous axonopathy caused by IDPN and the distal neurofilamentous axonopathies from n-hexane, acrylamide, and carbon disulfide intoxication. [14C]DMHD was found to alkylate nerve protein and to result in polymers of radiolabeled protein too large to pass through nitrocellulose filters with pore sizes as large as 12 nm. An even greater proportion of radiolabeled protein was retained by nitrocellulose filters when DMHD was reacted with nerve in which SCa (slow component a of axonal transport) had been pulse-labeled with [35S] methionine. Radiolabeled nerve proteins acylated with [125I]Bolton-Hunter reagent were minimally retained by nitrocellulose filters, suggesting that filter retention reflects polymerization rather than non-specific adsorption.
Acrylamide↗
The effect of 3,4-dimethyl substitution on the neurotoxicity of 2,5-hexanedione. I. Accelerated clinical neuropathy is accompanied by more proximal axonal swellings.
The neurotoxicity of the gamma-diketone, 3,4-dimethyl-2,5-hexanedione, was studied in rats and compared to the known neurotoxicity of the parent compound, 2,5-hexanedione. The test compound was found to be 20 to 30 times more potent on a molar basis than hexanedione. In addition, unlike the distal axonal changes associated with hexanedione, the neurofilamentous swellings following exposure to the dimethyl analog occurred more proximally in the axon, with a preponderance in the anterior horn and lateral tracts of the spinal cord, and in the anterior roots. Since alkyl substitution causes branched-chain compounds to cyclize more rapidly than unbranched analogs, the greater neurotoxicity of the dimethyl compound implicates pyrrole formation in the pathogenesis of n-hexane neuropathy. Furthermore, the location of the axonal swellings induced with 3,4-dimethyl 2,5-hexanedione suggests that there is a common mechanism of injury for the entire class of neurofilament neuropathies, providing a continuum between the intraspinal swellings of beta, beta'-iminodipropionitrile (IDPN) and the distal axonopathies of 2,5-hexanedione, carbon disulfide, and acrylamide. In addition, lower doses of 3,4-dimethyl-2,5-hexanedione for longer periods of time led to a shift in the location of the axonal swellings to include more distal sites. These observations support the hypothesis that covalent crosslinking of the stable neurofilament is the primary event in the molecular pathogenesis of these toxic neuropathies, and that the rate of crosslinking of neurofilaments determines the proximodistal location of the axonal swelling.
Animals↗
The effect of 3,4-dimethyl substitution on the neurotoxicity of 2,5-hexanedione. II. Dimethyl substitution accelerates pyrrole formation and protein crosslinking.
3,4-Dimethyl-2,5-hexanedione and 2,5-hexanedione were reacted with model amines to yield N-substituted 2,3,4,5-tetramethylpyrroles and 2,5-dimethylpyrroles, respectively. When compared to the unsubstituted parent compound 2,5-hexanedione, 3,4-dimethyl-2,5-hexanedione was found to cyclize approximately eight times as rapidly on a molar basis at 37 degrees C, with an activation energy of 3290 cal/mole less than 2,5-hexanedione. In addition, 1-benzyl-2,3,4,5-tetramethylpyrrole oxidized more readily than 1-benzyl-2,5-dimethylpyrrole with a difference in the half-wave potentials of 0.29 V. Both gamma-diketones led to progressive crosslinking of proteins in vitro, with the dimethyl substitution accelerating this process by a factor of 40. The formation of pyrrolyl derivatives in vivo was demonstrated by the characteristic absorption spectra obtained following reaction of erythrocyte proteins from intoxicated rats with Ehrlich's reagent. There was progressive formation of protein-bound dimethylpyrroles following exposure to 2,5-hexanedione and formation of tetramethylpyrroles following exposure to 3,4-dimethyl-2,5-hexanedione in vivo. Preparations of axonal pads also demonstrated pyrrole derivatization in vivo. In addition, spectrin preparations of erythrocytes from intoxicated rats showed a large amount of high molecular weight protein (400,000 Da), corresponding to dimerized spectrin. Thus, 3,4-dimethyl-2,5-hexanedione, which is 20 to 30 times more potent on a molar basis than 2,5-hexanedione in leading to a neurofilamentous neuropathy, is associated with more rapid pyrrole formation and protein crosslinking in vitro, and it has been demonstrated that these processes occur in vivo. These observations support the hypothesis that pyrrole formation and autoxidation occur following exposure to gamma-diketones, leading to covalent crosslinking of proteins in vivo, a process which may explain the pathogenesis of neurofilament accumulation in these neuropathies.
Animals↗
Studies of the molecular pathogenesis of hexane neuropathy. II. Evidence that pyrrole derivatization of lysyl residues leads to protein crosslinking.
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Cross-Linking Reagents↗