A variety of industrial and commercially-available chemicals cause a specific toxic neuropathy characterized by abnormal accumulation of neurofilaments within vulnerable axons. The widely used solvents n-hexane and methyl butyl ketone, which have been responsible for many instances of human occupational neurotoxic disease, are converted in vivo to 2,5-hexanedione (2,5-HD), a Gamma-diketone believed to interact directly with axonal components to induce the observed effects. This proposal is designed to evaluate the hypothesis that Gamma-diketones react with lysine Epsilon-amino groups of neurofilament (NF) and other axonal cytoskeletal proteins in vivo to form hydrophobic 2,5-dialkylpyrrole adducts, that this adduct formation is the critical event for induction of neuropathy, and that the resultant loss of critical lysine moieties and/or changes in protein hydrophobicity causes disruption of the specialized cytoskeletal protein transport mechanism within the axon and subsequent aggregation of NF protein. Since the altered protein persists within the axon and is unable to reach the nerve terminal for proteolysis, nutrient transport into the distal axon is interrupted and axonal degeneration occurs. Although pyrrole adduct formation occurs in non-neural proteins, clearance mechanisms effectively remove altered protein, preventing toxicity.
Specific aims designed to prove this hypothesis include quantitation of pyrrole adduct in neural and non-neural tissue protein from rats with prolonged oral exposure to 2,5-HD, and characterization of physico-chemical changes and molecular sites of binding within these proteins. Additional aims are examination of in vivo protein amine binding of non-neurotoxic 2,4-HD, assessment of the ability of this isomer to influence the time course of 2,5-HD neuropathy, and examination of the in vitro protein amine reactivity of the related neurotoxins carbon disulfide, acrylamide, and Beta,Beta'-iminodipropionitrile, with the long-range goal of elucidating a common mechanism of action for these compounds. Analytical techniques employed will include polyacrylamide gel electrophoresis, quantitative amino acid analysis, and analytical peptide mapping for identification of altered protein lysine moieties, and HPLC and sedimentation analysis of proteins for assessment of solubility changes and protein aggregation. Lysine reaction products will be characterized by mass spectroscopy, and tissue levels of 2,4- and 2,5-HD will be quantitated by gas chromatography. Results will provide substantial progress toward elucidation of the molecular mechanism of action of these important neurotoxic chemicals.
