Microtubule Assembly is Altered Following Covalent Modification by the n-Hexane Metabolite 2,5-Hexanedione

  • Kim Boekelheide
  • Julia Eveleth
  • M. Diana Neely
  • Tracy M. Sioussat
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 283)


2,5-Hexanedione (2,5-HD)1 is the ultimate toxic metabolite of aliphatic hexacarbon precursors such as n-hexane and methyl n-butyl ketone (Krasavage et al., 1980). 2,5-HD reacts with protein lysyl ε-amines to form pyrroles (DeCaprio et al., 1982; Graham et al., 1982; Anthony et al., 1983a,b; Genter et al., 1987). Pyrroles are unstable intermediates and undergo crosslinking reactions; the formation of protein crosslinks appears necessary for the development of testicular and nervous system toxicity (Boekelheide et al., 1988; St. Clair et al., 1988).


Sertoli Cell Microtubule Assembly Nucleation Time Sodium Glutamate Tubulin Assembly 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Abbreviations used






guanosine 5’-triphosphate




maximal velocity of assembly


time required to achieve maximal velocity of assembly

glutamate assembly buffer

1 M sodium glutamate, pH 6.60

Mes assembly buffer

0.1 M 2-[N-morpholino]ethanesulfonic acid, 1 mM EGTA, 1 mM MgCl2, pH 6.75


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Anthony, D.C., Boekelheide, K., Graham, D.G. (1983a). The effect of 3,4-dimethyl substitution on the neurotoxicity of 2,5-hexanedione. 1. Accelerated clinical neuropathy is accompanied by more proximal axonal swellings. Toxicol. Appt. Pharmacol. 71, 362–371.CrossRefGoogle Scholar
  2. Anthony, D.C., Boekelheide, K., Anderson, C.W., Graham, D.G. (1983b). The effect of 3,4-dimethyl substitution on the neurotoxicity of 2,5-hexanedione. II. Dimethyl substitution accelerates pyrrole formation and protein crosslinking. Toxicol. Appl. Pharmacol. 71, 372–382.CrossRefPubMedGoogle Scholar
  3. Arakawa, T., Timasheff, S.N. (1984). The mechanism of action of Na glutamate, lysine HC1, and piperazine-N,Ni-bis[2-ethanesulfonic acid] in the stabilization of tubulin and microtubule formation. J. Biol. Chem. 259, 4979–4986.PubMedGoogle Scholar
  4. Bergen, L.G., Borisy, G.G. (1983). Tubulin-colchicine complex inhibits microtubule elongation at both plus and minus ends. J. Biol. Chem. 258, 4190–4194.PubMedGoogle Scholar
  5. Boekelheide, K. (1987a). 2,5-Hexanedione alters microtubule assembly. I. Testicular atrophy, not nervous system toxicity, correlates with enhanced tubulin polymerization. Toxicol. Appl. Pharmacol. 88, 370–382.Google Scholar
  6. Boekelheide, K. (1987b). 2,5-Hexanedione alters microtubule assembly. II. Enhanced polymerization of crosslinked tubulin. Toxicol. App1. Pharmacol. 88, 383–396.Google Scholar
  7. Boekelheide, K. (1988a). Rat testis during 2,5-hexanedione intoxication and recovery. I. Dose response and the reversibility of germ cell loss. Toxicol. Appl. Pharmaco1. 92, 18–27.CrossRefGoogle Scholar
  8. Boekelheide, K. (1988b). Rat testis during 2,5-hexanedione intoxication and recovery. II. Dynamics of pyrrole reactivity, tubulin content and microtubule assembly. Toxicol. App1. Pharmacol. 92, 28–33.Google Scholar
  9. Boekelheide, K., Anthony, D.C., Giangaspero, F., Gottfried, M.R., Graham, D.G. (1988). Aliphatic diketones: influence of dicarbonyl spacing on amine reactivity and toxicity. Chem. Res. Toxicol. 1, 200–203.CrossRefPubMedGoogle Scholar
  10. Boekelheide, K., Eveleth, J. (1988). The rate of 2,5-hexanedione intoxication, not total dose, determines the extent of testicular injury and altered microtubule assembly in the rat. Toxicol. Appl. Pharmacol. 94, 76–83.CrossRefPubMedGoogle Scholar
  11. Boekelheide, K., Neely, M.D., Sioussat, T. (1989). The Sertoli cell cytoskeleton: A target for toxicant-induced germ cell loss. Toxicol. Appl. Pharmacol. 101, 373–389.CrossRefPubMedGoogle Scholar
  12. Chapin, R.E., Morgan, K.T., Bus, J.S. (1983). The morphogenesis of testicular degeneration induced in rats by orally administered 2,5-hexanedione. Exptl. Mot. Pathol. 38, 149–169.CrossRefGoogle Scholar
  13. Chapin, R.E., Norton, R.M., Popp, J.A., and Bus, J.S. (1982). The effects of 2,5- hexanedione on reproductive hormones and testicular enzyme activities in the F-344 rat. Toxicol. AppI. Pharmaco. 62, 262–272.CrossRefGoogle Scholar
  14. DeCaprio, A.P., Olajos, E.J., Weber, P. (1982). Covalent binding of a neurotoxic n-hexane metabolite: conversion of primary amines to substituted pyrrole adducts by 2,5-hexanedione. Toxicol. Appl. Pharmacol. 65, 440–450.CrossRefPubMedGoogle Scholar
  15. Demura, R., Suzuki, T., Nakamura, S., Komatsu, H., Jibiki, K., Odagiri, E., Demura, H., Shizume, K. (1987). Effect of uni-and bilateral cryptorchidism on testicular inhibin and testosterone secretion in rats. Endocricol. Japon. 34, 911–917.CrossRefGoogle Scholar
  16. Detrich, H.W., III, Jordan, M.A., Wilson, L., Williams, R.C., Jr. (1985). Mechanism of microtubule assembly. Changes in polymer structure and organization during assembly of sea urchin egg tubulin. J. Biol. Chem. 260, 9479–9490.PubMedGoogle Scholar
  17. GaleIla, G., Smith, D.B. (1982). The cross-linking of tubulin with imidoesters. Can. J. Biochem. 60, 71–80.CrossRefGoogle Scholar
  18. Garland, D.L. (1978). Kinetics and mechanism of colchicine binding to tubulin. Evidence for ligand-induced conformational change. Biochemistry 17, 4266–4272.CrossRefPubMedGoogle Scholar
  19. Gekko, K., Timasheff, S.N. (1981). Mechanism of protein stabilization by glycerol. Preferential hydration in glycerol-water mixtures. Biochemistry 20, 4667–4676.CrossRefPubMedGoogle Scholar
  20. Genter, M.B., Szakal-Quin, Gy., Anderson, C.W., Anthony, D.C., Graham, D.G. (1987). Evidence that pyrrole formation is a pathogenetic step in g-diketone neuropathy. Toxicol. Appl. Pharmacol. 87, 351–362.Google Scholar
  21. Graham, D.G., Anthony, D.C., Boekelheide, K., Maschmann, N.A., Richards, R.G., Wolfram, J.W., Shaw, B.R. (1982). Studies of the molecular pathogenesis of hexane neuropathy. II. Evidence that pyrrole derivatization of lysyl residues leads to protein crosslinking. Toxicol. Appl. Pharmacol. 64, 415–422.CrossRefPubMedGoogle Scholar
  22. Hagenas, L., Ritzen, E.M. (1976). Impaired Sertoli cell function in experimental cryptorchidism in the rat. Mo1. Cell. Endocrinol. 4, 25–34.CrossRefGoogle Scholar
  23. Hamel, E., Lin, C.M. (1981). Glutamate-induced polymerization of tubulin. Characterization of the reaction and application to the large-scale purification of tubulin. Arch. Biochem. Biophys. 209, 29–40.CrossRefPubMedGoogle Scholar
  24. Hardy, P.M., Hughes, G.J., Rydon, H.N. (1979). The nature of the cross-linking of proteins by glutaraldehyde. Part 2. The formation of quaternary pyridinium compounds by the action of glutaraldehyde on proteins and the identification of a 3-(2-piperidyl)-pyridinium derivative, anabilysine, as a cross-linking entity. J. Chem. Soc. Perkin I, 2282–2288.CrossRefGoogle Scholar
  25. Horowitz, P., Prasad, V., Luduena, R.F. (1984). Bis[1,8-anilinonaphthalenesulfonate]. A novel and potent inhibitor of microtubule assembly. J. Biol. Chem. 259, 14647–14650.PubMedGoogle Scholar
  26. Kerr, J.B., Rich, K.A., de Kretser, D.M. (1979). Effects of experimental cryptorchidism on the ultrastructure and function of the Sertoli cell and peritubular tissue of the rat testis. Biol. Reprod. 21, 823–838.CrossRefPubMedGoogle Scholar
  27. Krasavage, W.J., O’Donoghue, J.L., DiVincenzo, G., Terhaar, C.J. (1980). The relative neurotoxicity of methyl n-butyl ketone, n-hexane and their metabolites. Toxicol. Appl. Pharmacol. 52, 433–441.CrossRefPubMedGoogle Scholar
  28. Lee, J.C., Timasheff, S.N. (1977). In vitro reconstitution of calf brain microtubules. Effects of solution variables. Biochemistry 16, 1754–1764.Google Scholar
  29. Neely, M.D., Boekelheide, K. (1988). Sertoli cell processes have axoplasmic features: an ordered microtubule distribution and an abundant high molecular weight microtubule associated protein (cytoplasmic dynein). J. Cell Biol. 107, 1767–1776.CrossRefPubMedGoogle Scholar
  30. Nelson, W.O. (1951). Mammalian spermatogenesis: effects of experimental cryptorchidism in the rat and non-descent of the testis in man. Recent Prog. Horm. Res. 6, 29–62.Google Scholar
  31. Nishimune, Y., Aizawa, S. (1978). Temperature sensitivity of DNA synthesis in mouse testicular germ cells in vitro. Exp. Cell Res. 113, 403–408.CrossRefGoogle Scholar
  32. Prasad, A.R.S., Luduena, R.F., Horowitz, P.M. (1986). Bis[8-anilinonaphthalene-1sulfonate] as a probe for tubulin decay. Biochemistry 25, 739–742.CrossRefPubMedGoogle Scholar
  33. Russell, L.D., Malone, J.P., MacCurdy, D.S. (1981). Effect of the microtubule disrupting agents, colchicine, and vinblastine, on seminiferous tubule structure in the rat. Tissue Cell 13, 349–367.CrossRefPubMedGoogle Scholar
  34. St. Clair, M.B.G., Amarnath, V., Moody, A., Anthony, D.C., Anderson, C.W., Graham, D.G. (1988). Pyrrole oxidation and protein cross-linking as necessary steps in the development of g-diketone neuropathy. Chem. Res. Toxicol. 1, 179–185.Google Scholar
  35. Shelanski, M.L., Gaskin, F., Cantor, C.R. (1973). Microtubule assembly in the absence of added nucleotides. Proc. Nat. Acad. Sci. USA 70, 765–768.CrossRefPubMedGoogle Scholar
  36. Sioussat, T., Boekelheide, K. (1989). Selection of a nucleation-promoting element following chemical modification of tubulin. Biochemistry 28, 4435–4443.CrossRefPubMedGoogle Scholar
  37. Szasz, J., Burns, R., Sternlicht, H. (1982). Effects of reductive methylation in microtubule assembly. J. Biol. Chem. 257, 3697–3704.PubMedGoogle Scholar
  38. Vogl, A.W., Lin, Y.C., Dym, M., Fawcett, D.W. (1983a). Sertoli cells of the golden-mantled ground squirrel (Spermophilus lateralis): a model system for the study of shape change. Am. J. Anat. 168, 83–98.Google Scholar
  39. Vogl, A.W., Linck, R.W., Dym, M. (1983b). Colchicine-induced changes in the cytoskeleton of the golden-mantled ground squirrel (Spermophilus lateralis) Sertoli cells. Am. J. Anat. 168, 99–108.CrossRefPubMedGoogle Scholar
  40. Voter, W.A., Erickson, H.P. (1984). The kinetics of microtubule assembly. Evidence for a two-stage nucleation mechanism. J. Biol. Chem. 259, 10430–10438.PubMedGoogle Scholar
  41. Weisenberg, R.C. (1972). Microtubule formation in vitro in solutions containing low calcium concentrations. Science 177, 1104–1105.CrossRefPubMedGoogle Scholar

Copyright information

© Plenum Press, New York 1991

Authors and Affiliations

  • Kim Boekelheide
    • 1
  • Julia Eveleth
    • 1
  • M. Diana Neely
    • 1
  • Tracy M. Sioussat
    • 1
  1. 1.Department of Pathology and Laboratory MedicineBrown UniversityProvidenceUSA

Personalised recommendations