CS2-MEDIATED CROSS-LINKING OF ERYTHROCYTE SPECTRIN AND NEUROFILAMENT PROTEIN - DOSE-RESPONSE AND TEMPORAL RELATIONSHIP TO THE FORMATION OF AXONAL SWELLINGS

Using model proteins, a mechanism for CS2-mediated covalent cross-link ing of proteins has been demonstrated previously, The biologic importa nce of CS2-promoted protein cross-linking is apparent as a possible do simeter of CS2 exposure and as a potential mechanism to account for th e identical neuropathies produced by 2,5-hexanedione and CS2. The pres ent investigation examines the utility of erythrocyte spectrin cross-l inking as a biomarker of effect for inhalation exposure to CS2 and exa mines the ability of CS2 to cross-link neurofilament proteins, a poten tial neurotoxic target. Rats were exposed to CS2 via inhalation at con trol, 50-, 500-, and 800-ppm levels for 2, 4, 8, and 13 weeks and spec trin dimer formation was quantified using denaturing gel electrophores is and densitometry. Neurofilament preparations were also obtained fro m spinal cords and examined for cross-linking using Western blotting m ethods, The results obtained for protein crosslinking were compared to morphologic changes in the cervical and lumbar spinal cord using ligh t and electron microscopy. The spectrin dimer exhibited a cumulative d ose response and was detectable at both the 50-ppm level employed that did not produce axonal swellings and prior to the development of axon al swellings for the 500- and 800-ppm levels used. Neurofilament prote in cross-linking involved all three subunits and the temporal relation ship of cross-linking was consistent with a contributing role in the d evelopment of axonal swellings. These results establish the sensitivit y of spectrin cross-linking for evaluating inhalation exposures and ex tend the similarities observed for 2,5-hexanedione and CS2 in both cli nical settings and in vitro models to their effects exerted on neurofi laments in the axon. (C) 1997 Academic Press.