Effects of 2,5-hexanedione on calpain-mediated degradation of human neurofilaments in vitro.

2,5-Hexanedione (2,5-HD), the neurotoxic metabolite of n-hexane, can structurally modify neurofilaments (NF) by pyrrole adduct formation and subsequent covalent cross-linking. 2,5-HD also induces accumulations of NF within the pre-terminal axon. We examined whether exposure of NF to 2,5-HD affected NF degradation. Two different models were used: (1) NF-enriched cytoskeletons isolated from human sciatic nerve were incubated with 2,5-HD in vitro and (2) differentiated human neuroblastoma cells (SK-N-SH) were exposed to 2, 5-HD in culture prior to isolation of cytoskeletal proteins. The cytoskeletal preparations were subsequently incubated with calpain II. The amount of NF-H and NF-L remaining after proteolysis was determined by SDS-PAGE and quantitative immunoblotting. NF-M proteolysis could not be quantified. Incubation of sciatic nerve cytoskeletal preparations with 2,5-HD resulted in cross-linking of all three NF proteins into high molecular weight (HMW) material with a range of molecular weights. Proteolysis of the NF-H and NF-L polypeptides was not affected by 2,5-HD-exposure. Degradation of the HMW material containing NF-H or NF-L was retarded when comparing with degradation of the NF-H and NF-L polypeptides, respectively, from control samples, but not as compared to the corresponding NF polypeptides from 2,5-HD-treated samples. Exposure of SK-N-SH cells to 2,5-HD also resulted in considerable cross-linking of NF. No differences were found between the proteolytic rates of NF-L and NF-H from exposed cells as compared with those subunits from control cells. Moreover, degradation of cross-linked NF-H was not different from monomeric NF-H. In conclusion, whether 2,5-HD affects calpain-mediated degradation of cross-linked NF proteins will depend on which model better reflects NF cross-linking as occurring in 2, 5-HD-induced axonopathy. However, with both models it was demonstrated that exposure of NF proteins to 2,5-HD without subsequent cross-linking is not adequate to inhibit NF proteolysis in vitro by added calpain.

Sensitivity to 2,5-hexanedione of neurofilaments in neuroblastoma cell line SK-N-SH increases during differentiation.

The effect of 2,5-hexanedione (2,5-HD) on the distribution of the neurofilamental (NF) proteins and vimentin was examined in human neuroblastoma cell line SK-N-SH with immunocytochemical methods. Retinoic acid (10 microM) induced differentiation into neuronal cells resulting in the outgrowth of processes and synthesis of NF proteins in the majority of the cells. A minority (4%) differentiated as large fibroblasts. Cells were exposed to 0-10 mM 2,5-HD for 3 days. In neuronal cells a concentration-dependent accumulation of NF proteins was detected as a spherical structure in the perikaryon. Neurofilaments in differentiated SK-N-SH cells were more susceptible to 2,5-HD than NF in undifferentiated cells, as the effects were observed at much lower 2,5-HD concentrations. In contrast, no accumulation of vimentin was detected in the fibroblastic cells.

Metabolic activation of halothane to neoantigens in C57Bl/10 mice: immunochemical studies.

C57Bl/10 mice were given halothane (10 mmol/kg, intraperitoneally) and microsomal proteins were analysed for the presence of trifluoroacetylated (TFA) neoantigens by SDS-gel electrophoresis followed by immunoblotting using a polyclonal anti-TFA antibody. In microsomal preparations from liver, lung and olfactory tissues, a 54 kDa TFA adduct was detectable 1 h after dosing. After 3-48 h, multiple bands were detected in liver (45-100 kDa) and in the lung (26-57 kDa) and in one experiment in which [14C]halothane was given, several immunoreactive bands from liver microsomes were shown to contain a covalently bound metabolite of the drug. In olfactory tissue, initially (1 h), a major band of 54 kDa and a less prominent component of about 50 kDa were seen. The number of bands increased at later times but the additional bands were far fewer than in liver. The rate of decay of the 54 kDa adduct was also measured in both liver and olfactory microsomes and found to be compatible with the reported turnover of total liver cytochrome P-450. 24 h after treating mice with halothane (10 mmol/kg), no TFA neoantigens could be detected on the outer cell surface of isolated viable hepatocytes when analysed by fluorescence activated flow cytometry. In contrast, non-viable cells, or those fixed in acetone were all positive. Using immunohistochemistry, TFA neoantigens were demonstrated in the centrilobular area of the liver, the non-ciliated bronchiolar epithelial (Clara) cells of the lung, proximal tubular cells of the kidney and the respiratory and olfactory epithelium of nasal tissues.

Effect of aluminum chloride, -citrate, and -maltol on the calcium-mediated degradation of neurofilament proteins.

Aluminum (Al) has been observed to cause neurofilament protein accumulation in both experimental animals and cultured cells. Impairment of axonal transport is thought to be a mechanism of toxicity. Inhibition of the degradation of neurofilament proteins, however, resulting in accumulation of these proteins may be an alternative mechanism for Al toxicity. In the present study, the effect of calcium (Ca) on the proteolysis of the neurofilament triplet proteins by calcium-activated neutral proteases (CANP) was studied in the isolated sciatic nerve explants. The extent of the degradation was found to be dependent on the Ca concentration. The effect of Al chloride, -citrate and -maltol on the calcium-induced degradation was studied. No effect of any of the Al compounds was observed, suggesting that the metal may exert its neurotoxic effect via a mechanism other than impairment of neurofilament proteolysis. Maltol itself was found to enhance the effect of Ca on the degradation of neurofilament proteins, probably by facilitating the movement of Ca across the neuronal membrane.

Accumulation of aluminium in rat liver: association with constituents of the cytosol.

Aluminium (Al) accumulation occurs in the liver of renal patients and in patients on parenteral nutrition. Human hepatotoxicity is not proven. The role of the liver in storage and biotransformation of Al and in development of osteo- and neurotoxicity is not clarified as yet. The aim of the present investigation was to study the storage of Al in total liver and in subcellular liver fractions, and its association with soluble cytosolic molecular species. Therefore, rats were loaded with Al prior to liver fractionation by ultracentrifugation, and equilibrium gel filtration chromatography of the cytosol, using a previously described method for Al speciation in serum. Al accumulated dose-dependently in liver and subcellular liver fractions, the lowest levels occurring in the cytosol. A dose-dependent elevation of Al in the blood was also observed. Gelfiltration of the cytosol indicated that Al was associated with a low molecular weight form which was not a citrate complex, and a high molecular weight form, which was larger than transferrin. No induction of and association with metallothionein occurred.