Alterations in rat axonal cytoskeletal proteins induced by in vitro and in vivo 2,5-hexanedione exposure.

The neurotoxic gamma-diketone 2,5-hexanedione (2,5-HD) reacts in vitro and in vivo with protein lysine epsilon-amine moieties to yield 2,5-dimethylpyrrole adducts. It has been hypothesized that pyrrole adduct formation in neurofilament (NF) or other axonal proteins may lead to increased hydrophobicity, secondary autoxidative crosslinking, or the loss of essential lysine amine groups, and that pyrrolylation therefore represents the critical initiating event in gamma-diketone neuropathy. The present investigation was designed to evaluate pyrrole levels and other changes in brain stem and spinal cord axonal cytoskeletal proteins from rats receiving 0.5% 2,5-HD in the drinking water for up to 8 weeks and following recovery. Clinical signs of neuropathy were apparent in rats after 5 weeks exposure, while no histopathological effects were seen until 8 weeks. Cessation of dosing resulted in some recovery from clinical neuropathy but virtually no change in histopathologically demonstrable CNS damage. 2,5-Dimethylpyrrole adduct was detected in serum and axonal cytoskeletal proteins from animals in all exposure groups and its formation appeared to reach a plateau in both serum and axonal protein. Assay of total protein lysine vs pyrrole content demonstrated an average conversion of less than 1% of epsilon-amine groups into pyrrole adducts in axonal protein after 2 weeks exposure. Gel electrophoresis revealed discrete new protein bands in brain stem and spinal cord axonal protein preparations from treated animals, along with high-molecular-weight, nonmigrating proteinaceous material. Concentration of the nonmigrating material appeared to increase in a time-dependent fashion. A concurrent decrease in the relative amounts of native NF subunit proteins was observed in brain stem but not spinal cord. Reversal of these changes was observed 9 weeks after cessation of dosing, although residual nonmigrating protein and pyrrole adduct were present. In vitro incubation of axonal cytoskeletal protein preparations (pH 7.2, 37 degrees C) with 2,5-HD resulted in the formation of high-molecular-weight bands identical to those seen in vivo. These findings provide evidence for pyrrole adduct formation and secondary covalent crosslinking in CNS axonal cytoskeletal proteins from 2,5-HD-treated animals.