Superfund Research Program

Biomarkers of Neurotoxicant Exposure and Neurodegeneration

Project Leaders: Mohammad I. Sabri, Peter S. Spencer
Grant Number: P42ES010338
Funding Period: 2000-2006

Project-Specific Links

Final Progress Reports

Year:   2005 

Research Problem: Superfund sites, such as Oregon’s Portland Harbor, are contaminated with aromatic and aliphatic hydrocarbon solvents with neurotoxic potential. Drs Sabri and Spencer seek to understand how the gamma-diketone metabolites of these solvents elicit neurotoxicity. Then hypothesize that these gamma-diketones react with vital neural proteins and form polymeric adducts that may be involved in causing axon degeneration in the central (CNS) and peripheral (PNS) nervous system. Test agents include the neurotoxic metabolite of the aliphatic hydrocarbon solvent n-hexane, 2,5-hexanedione (2,5-HD) and the aromatic metabolite of 1,2-diethylbenzene,1,2-diacetylbenzene (1,2-DAB) together with their corresponding non-protein-reactive, non-neurotoxic isomers, 2,4-hexanedione (2,4-HD) and 1,3-diacetylbenzene (1,3-DAB), respectively.

Accomplishments: The researchers have established, in mice, the relationship between the protein-reactivity, chromogenicity and neurotoxicity of 1,2-DAB relative to its non-neurotoxic isomer 1,3-DAB. The estimated neurotoxic potency of 1,2-DAB:2,5-HD is ~ 30:1. Gamma diketones react with the epsilon amino groups of lysine residues, and therefore their protein reactivity correlates with total lysine content. Thus lysine-rich NF-H and NF-M are more susceptible than lysine-poor NFL and beta-tubulin to 1,2-DAB. The research team’s in vitro and in vivo studies have shown that motor proteins kinesin and dynein and microtubule-associated protein tau of rat spinal cord react with 1,2-DAB in proportion to their lysine content (kinesin > dynein = tau). Mapping 2,5-HD-adducted NF proteins (NFP) depends on the sites of posttranslational modification of neurofilament proteins (NFP). The extensive phosphorylation of NFPs appears to play a significant role in stabilizing the 2,5-HD-adducted NFP.

High-resolution global proteomics analysis by cL-FTICR-mass spectrometry of the brain cytosol of mice treated with a single dose of 1,2-DAB or 1,3-DAB showed large increases for stathmin (~ 8 fold in 1,2-DAB relative to 1,3-DAB) one week later. Mapping data to metaCore/MetaDrug functional analysis software (Gene Go) revealed the increased presence of stathmin and related proteins uniquely after 1,2-DAB treatment. These findings are of importance because stathmin gene knockout mice develop an age-dependent CNS/PNS axonopathy with many similarities to 1,2-DAB-induced axonopathy.

Primary hippocampal neuronal cultures are being used to develop a model of aromatic gamma-diketone neurotoxicity in vitro. Micrographs of living hippocampal neurons treated with DAB isomers show that neurotoxic 1,2-DAB, but not 1,3-DAB, induce concentration- and time-dependent axonal pathology.

Significance:  The major observation is that certain aromatic hydrocarbons have the potential to induce peripheral neuropathy because they produce gamma-diketone metabolites that react with neuroproteins. This was previously established for straight chain compounds (n-hexane, 2-hexanone) but not for benzene derivatives. The specific targets of gamma-diketones are important quests because their identification will likely lead to an understanding of molecular mechanisms underlying neuropathy, a common disorder of the nervous system associated with prolonged exposure to selected environmental pollutants and drugs.