Toxic neuropathies: Mechanistic insights based on a chemical perspective

Abstract

2,5-Hexanedione (HD) and acrylamide (ACR) are considered to be prototypical among chemical toxicants that cause central-peripheral axonopathies characterized by distal axon swelling and degeneration. Because the demise of distal regions was assumed to be causally related to the onset of neurotoxicity, substantial effort was devoted to deciphering the respective mechanisms. Continued research, however, revealed that expression of the presumed hallmark morphological features was dependent upon the daily rate of toxicant exposure. Indeed, many studies reported that the corresponding axonopathic changes were late developing effects that occurred independent of behavioral and/or functional neurotoxicity. This suggested that the toxic axonopathy classification might be based on epiphenomena related to dose-rate. Therefore, the goal of this mini-review is to discuss how quantitative morphometric analyses and the establishment of dose-dependent relationships helped distinguish primary, mechanistically relevant toxicant effects from non-specific consequences. Perhaps more importantly, we will discuss how knowledge of neurotoxicant chemical nature can guide molecular-level research toward a better, more rational understanding of mechanism. Our discussion will focus on HD, the neurotoxic γ-diketone metabolite of the industrial solvents n-hexane and methyl-n-butyl ketone. Early investigations suggested that HD caused giant neurofilamentous axonal swellings and eventual degeneration in CNS and PNS. However, as our review will point out, this interpretation underwent several iterations as the understanding of γ-diketone chemistry improved and more quantitative experimental approaches were implemented. The chemical concepts and design strategies discussed in this mini-review are broadly applicable to the mechanistic studies of other chemicals (e.g., n-propyl bromine, methyl methacrylate) that cause toxic neuropathies.

Keywords: 2,5-Hexanedione; Acrylamide; Central–peripheral distal axonopathy; Dying back neuropathy; Neurotoxicity; Peripheral neuropathy.

Figure 1

Cross-sectional areas from (A) spinal cord gracile regions and (C) distal tibial nerve from age-matched controls and moderately affected rats intoxicated at either 175 mg/kg/d × 84 days or 400 mg/kg/d × 17 days. Also presented are corresponding percentage frequency distributions of cross-sectional areas (µM²) for (B) gracile regions and (D) distal tibial nerve from control and HD-intoxicated rats. For each frequency distribution, corresponding percentile data (µM²) are presented and the horizontal lines represent the upper limit (100^th percentile) for the control distributions. Results indicate that, regardless of HD dose-rate, the respective frequency graphs are shifted to the left relative to control distributions. This shift toward smaller diameters is reflected by significant decreases in the corresponding 25^th, 50^th and 75^th percentiles (insert). These data indicate that regardless of dose-rate, axon atrophy developed in gracile and tibial nerve regions was an early consequence of HD intoxication. In both regions, however, giant neurofilamentous swellings were prevalent only in severely intoxicated animals; i.e., 175 mg/kg/d × 98 days and 400 mg/kg/d × 21 days [4, 13, 16].

Figure 2

Representative immunoblots are presented for NF proteins (NFH, NFL and NFM) in co-sedimentation preparations of spinal cord form control and HD-intoxicated (400 and 175 mg/kg/d) rats (Zhang et al. [27]). Separation of proteins in denaturing conditions (tris-acetate/SDS-PAGE) revealed detergent-insoluble HMW bands for all NF subunit proteins in both control and HD samples. These HMW species were present but not prevalent in control as evidenced by the fact that they were detected only when control protein concentrations were higher (25 µg) than the concentrations used for HD samples (5 µg). Furthermore, cross-comparisons among NF proteins from control samples relevaled sevral HMW NFL bands (e.g., 280 kDa, 364 kDa) that were common to both NFH and NFM (see connecting arrows).