Peripheral nerve and brain differ in their capacity to resolve N,N-diethyldithiocarbamate-mediated elevations in copper and oxidative injury

Abstract

Previous studies have demonstrated that N,N-diethyldithiocarbamate (DEDC) elevates copper and promotes oxidative stress within the nervous system. However, whether these effects resolve following cessation of exposure or have the potential to persist and result in cumulative injury has not been determined. In this study, an established model for DEDC myelin injury in the rat was used to determine whether copper levels, oxidative stress, and neuromuscular deficits resolve following the cessation of DEDC exposure. Rats were exposed to DEDC for 8 weeks and then either euthanized or maintained for 2, 6 or 12 weeks after cessation of exposure. At each time point copper levels were measured by inductively coupled mass spectrometry to assess the ability of sciatic nerve, brain, spinal cord and liver to eliminate excess copper post-exposure. The protein expression levels of glutathione transferase alpha, heme oxygenase 1 and superoxide dismutase 1 in peripheral nerve and brain were also determined by western blot to assess levels of oxidative stress as a function of post-exposure duration. As an initial assessment of the bioavailability of the excess copper in brain the protein expression levels of copper chaperone for superoxide dismutase 1, and prion protein were determined by western blot as a function of exposure and post-exposure duration. Neuromuscular function in peripheral nerve was evaluated using grip strengths, nerve conduction velocities, and morphologic changes at the light microscope level. The data demonstrated that in peripheral nerve, copper levels and oxidative stress return to control levels within several weeks after cessation of exposure. Neuromuscular function also showed a trend towards pre-exposure values, although the resolution of myelin lesions was more delayed. In contrast, total copper and antioxidant enzyme levels remained significantly elevated in brain for longer post-exposure periods. The persistence of effects observed in brain suggests that the central nervous system is more susceptible to long-term cumulative adverse effects from dithiocarbamates. Additionally, significant changes in expression levels of chaperone for superoxide dismutase 1, and prion protein were observed consistent with at least a portion of the excess copper being bioactive.

Figure 1

Total tissue copper levels. Values for brain (closed squares), spinal cord (open circles), liver (open squares) and tibial nerve (closed triangles) were determined by ICP-MS (n=4, except n=12 for controls and n=6 for 8 week DEDC exposure group) and are reported as mean ppm dry weight of tissue. Error bars represent SE. **p <0.01 relative to controls by one-way ANOVA and Dunnett’s Multiple Comparison post hoc test.

Figure 2

GST-α, HO-1 and SOD1 protein expression in sciatic nerve and brain. (A) Representative western blot showing relative amounts of GST-α in proteins isolated from the brains of animals at the 8 week DEDC exposure time point as compared to controls. (B) Levels of GST-α in brain (open circles) and sciatic nerve (closed circles) determined by western blot. Optical density of GST-α was normalized to that of actin within the same sample and then to the mean value obtained for control samples on the same membrane (n =4 for controls and n=6 for exposed at 8 weeks; n=6 for controls and n=4 for exposed at all other time points). (C) Levels of HO-1 in brain (open circles) and sciatic nerve (closed circles) determined by western blot. Optical density determinations and exposure groups were identical to those used in (B) for GST-α. (D) Levels of SOD1 in brain (open circles) and sciatic nerve (closed circles) determined by western blot. Optical density determinations and exposure groups were identical to those used in (B) for GST-α. Error bars represent SE. ** p < 0.01 and * p < 0.05 relative to controls run on the same membrane by one-way unpaired students t-test.

Figure 3

CCS, and prion protein expression in brain as a function of DEDC exposure and post exposure duration. (A) Levels of CCS protein in brain determined by western blot. Optical density of CCS was normalized to that of actin within the same sample and then to the mean value obtained for control samples on the same membrane (n =4 for controls and n=6 for exposed at 8 weeks; n=6 for controls and n=4 for exposed at all other time points). (B) Levels of prion protein in brain determined by western blot. Optical density determinations and exposure groups were identical to those used in (A) for CCS. Error bars represent SE. **p <0.01 and * p <0.05 relative to controls run on the same membrane by one-way unpaired students t-test..

Figure 4

Body weight gain, hind limb grip strength and nerve conduction velocities. (A) Body weight gain of DEDC exposed animals presented as percent of time matched controls. (For controls, n=12 at 0 and 8 weeks and n=6 at 10, 14 and 20 weeks; for the DEDC exposed n= 18 at 8 weeks, n=12 at 10 weeks, n=8 at 14 weeks and n=4 at 20 weeks). **p < 0.01 and *p < 0.05 relative to time matched controls by one-way unpaired t-test. (B) Values for grip strengths from controls (0 weeks exposure) and DEDC exposed animals are reported as peak compression (grams) ± SE (n=4 except n = 12 for controls and n=6 for 8 week exposure group). (C) Values for NCV are reported as mean velocity (m/s) ± SE. (n=4 except n = 11 for controls (0 weeks exposure) and n=6 for 8 week exposure group). **p < 0.01 relative to control groups by one-way ANOVA and Dunnett’s Multiple Comparison post hoc test for grip strengths and NVC measurements.

Figure 5

Morphology of sciatic nerve cross-sections stained with toluidine blue. (A) Cross section obtained from a control rat showing the presence of large and small myelinated axons. The axons are surrounded by normal compact myelin with some axons sectioned through Schmidt-Lanterman incisures (white arrow). (B) Cross section of a sciatic nerve from an animal 2 weeks post DEDC exposure demonstrating an axon with intramyelinic edema (black arrow), a demyelinated axon (white arrow) surrounded by Schwann cell cytoplasm, and a degenerating axon (black arrowhead). (C) Cross section obtained from a rat 12 weeks post DEDC exposure showing two axons with thin myelin (white arrows) and myelin debris in the cytoplasm of a Schwann cell (black arrow)