Doyle G. Graham, MD PhD - US grants

Studies in this laboratory have developed the following hypothesis for the pathogenesis of the neurotoxicology of n-hexane, an environmental toxin of petroleum origin and a hazard of occupational exposure and inhalant drug abuse, viz., hexane is metabolized by the liver to the Gamma-diketone, 2,5-hexanedione, which reacts with amino groups of proteins resulting in an imine, which then cyclizes to form 2,5-dimethylpyrrole derivatives; autoxidation of pyrrole rings rendering them vulnerable to nucleophilic attack, resulting in covalent crosslinking of proteins; progressive crosslinking of neurofilaments during slow axonal transport generates aggregates too large to pass through nodes of Ranvier; the ensuing obstruction of slow axonal transport results in degeneration of the distal axon. This hypothesis is based on our observations that 2,5-hexanedione results in extensive pyrrole derivatization and protein crosslinking in vitro and in vivo. To test this hypothesis we shall (1) administer monoketones to rats to determine the relationships between diketone synthesis, diketone structure, clinical neurotoxicology, paranodal neurofilament accumulation and axonal degeneration; (2) characterize the kinetics of reaction of Gamma-diketones with primary amines and with lysyl groups of neurofilaments and other proteins; (3) establish proof of structure of the protein-protein crosslinks through model compound synthesis, (4) define whether reaction with neurofilaments results in aggregation through covalent crosslinking or via hydrophobic interactions; (5) seek morphologic, autoradiographic, and gel electrophoretic evidence for progressive neurofilament crosslinking by Gamma-diketones during slow axonal transport and obstruction of transport at nodes of Ranvier; and (6) determine whether additional environmental toxins which result in neurofilament accumulation do so via covalent crosslinking. Understanding the mechanism of neurofilament accumulation in hexane toxicity will direct prevention and therapy and allow generalizations to be made to other environmental toxins and exposure limited before human cases ever develop.

The proposal seeks to continue studies to define the molecular pathogenesis of the neuropathy caused by n-hexane, a hazard of occupational exposure and inhalant abuse. The working hypothesis guiding this work has been that n-hexane is metabolized to the gamma-diketone, 2,5-hexanedione, which reacts with lysyl amino groups of proteins to form pyrrolyl derivatives; the pyrrole rings then undergo autoxidation, resulting in covalent crosslinking of proteins; the extreme stability of the neurofilament predisposes this axonal filament to progressive derivatization and crosslinking; periodic constrictions of the axon at nodes of Ranvier present obstructions to the proximo-distal transport of the growing mass of neurofilaments; continued transport up to the point of occlusion results in large paranodal swellings filled with neurofilaments; subsequent impairment of axonal transport results in degeneration of the distal axon. To test this hypothesis the following. Specific Aims shall be pursued: (1) to determine the specificity and generality of the gamma-diketone structure in the genesis of the neurotoxicity of alkanes; (2) to define the mechanism of pyrrole synthesis and the relationship between the rate of pyrrole formation and neurotoxicity; (3) to determine whether pyrrole autoxidation and crosslinking are necessary steps in the pathogenetic sequence: (4) to determine the structure of the crosslinking adducts; (5) to determine whether neurofilamentous crosslinking is progressive during chronic intoxication; (6) to determine the role of axonal constrictions at nodes of Ranvier in the development of axonal swellings; (7) to determine the mechanism of degeneration of the distal axon; and (8) to determine what steps in the proposed pathogenetic scheme for n-hexane neuropathy have parallels in the pathogenesis of the neuropathies caused by beta,beta'-iminodipropionitrile, carbon disulfide and acrylamide. The major approach taken in this project is to synthesize novel analogues of 2,5-hexanedione and the putative crosslinking metabolites of other toxicants in order to test specific steps in the pathogenetic schemes and to define the identities of the crosslinking adducts. This includes the synthesis of a series of gamma-diketones which, through the presence of electron-withdrawing or electron-donating groups, enhance or impair the rate of pyrrole formation and retard or facilitate oxidation of the resulting pyrrole ring. Further, the role of oxidation of intermediates in the crosslinking of neurofilaments by beta,beta'- iminodipropionitrile and carbon disulfide will be explored. The participation of different neurofilament subunits in the crosslinking reactions will be sought as will the role of fast axonal transport in the genesis of distal axonal degeneration.