Previous studies from this laboratory have demonstrated that the addition of methyl groups at the 3 and 4 positions of the
2,5-hexanedione (2,5-HD) molecule results both in more rapid
pyrrole formation and in enhanced neurotoxicity. In order to define more clearly the relationship between rates of
pyrrole formation and neurotoxicity, the dl and meso diastereomers of
3,4-dimethyl-2,5-hexanedione (DMHD),
3,4-diethyl-2,5-hexanedione (
DEHD), and
3,4-diisopropyl-2,5-hexanedione (
DiPHD) were synthesized and purified. The rates of
pyrrole formation were compared with that of unsubstituted 2,5-HD, and rates of in vitro crosslinking were determined. Each of the compounds was administered to rats to determine relative neurotoxicity. Hindlimb
paralysis was reached after a total administered dose of 1.6 mmol/kg of dl-DMHD, while 5.9 mmol/kg of meso-DMHD was required.
Paralysis was not achieved with either diastereomer of
DEHD or
DiPHD, although both produced systemic toxicity. Histologic sections of spinal cords and anterior roots from rats treated with DMHD revealed large neurofilament-filled axonal swellings, while more distal sections contained axons undergoing Wallerian-type degeneration. Neither axonal swellings nor Wallerian-type degeneration were seen in sections from spinal cord or peripheral nerve of rats treated with
DEHD or
DiPHD. The rates of
pyrrole formation were in the order dl-DMHD greater than meso-DMHD greater than 2,5-HD greater than dl-
DEHD greater than meso-
DEHD greater than dl-
DiPHD greater than meso-
DiPHD, while in vitro crosslinking rates were in the order dl-DMHD greater than meso-DMHD greater than dl-
DEHD greater than meso-
DEHD greater than 2,5-HD greater than dl-
DiPHD greater than meso-
DiPHD. Cyclic voltammetry showed that the autoxidation of
pyrroles derived from DMHD,
DEHD, and
DiPHD occurred more readily than that derived from 2,5-HD. In addition, we report for the first time the segregation of axoplasmic organelles in animals treated with DMHD, providing further evidence that the neurofilamentous axonopathies caused by such compounds as
beta,beta'-iminodipropionitrile (IDPN), 2,5-HD and CS2 share a common underlying mechanism. The strong correlations between rates of
pyrrole formation, rates of in vitro crosslinking and relative neurotoxicity are seen as evidence that
pyrrole formation is a step in the pathogenetic sequence of gamma-diketone neuropathy.