Copper accumulation and lipid oxidation precede inflammation and myelin lesions in N,N-diethyldithiocarbamate peripheral myelinopathy

Abstract

Dithiocarbamates have a wide spectrum of applications in industry, agriculture and medicine with new applications being actively investigated. One adverse effect of dithiocarbamates is the neurotoxicity observed in humans and experimental animals. Results from previous studies have suggested that dithiocarbamates elevate copper and promote lipid oxidation within myelin membranes. In the current study, copper levels, lipid oxidation, protein oxidative damage and markers of inflammation were monitored as a function of N,N-diethyldithiocarbamate (DEDC) exposure duration in an established model for DEDC-mediated myelinopathy in the rat. Intra-abdominal administration of DEDC was performed using osmotic pumps for periods of 2, 4, and 8 weeks. Metals in brain, liver and tibial nerve were measured using ICP-MS and lipid oxidation assessed through HPLC measurement of malondialdehyde in tibial nerve, and GC/MS measurement of F(2) isoprostanes in sciatic nerve. Protein oxidative injury of sciatic nerve proteins was evaluated through quantification of 4-hydroxynonenal protein adducts using immunoassay, and inflammation monitored by quantifying levels of IgGs and activated macrophages using immunoassay and immunohistochemistry methods, respectively. Changes in these parameters were then correlated to the onset of structural lesions, determined by light and electron microscopy, to delineate the temporal relationship of copper accumulation and oxidative stress in peripheral nerve to the onset of myelin lesions. The data provide evidence that DEDC mediates lipid oxidation and elevation of total copper in peripheral nerve well before myelin lesions or activated macrophages are evident. This relationship is consistent with copper-mediated oxidative stress contributing to the myelinopathy.

Figure 1

Copper levels in posterior tibial nerve, liver and brain for controls (0-week) and each DEDC exposure duration (n=4, except for DEDC 8-week n=5) were determined by ICP-MS. Values are means ± SEM., and reported as ppm dry weight of nerve. * p < 0.05, **p < 0.01 and ***p < 0.001, One-way ANOVA and Tukey-Kramer Multiple Comparisons Test as compared to zero time point.

Figure 2

Lipid oxidation products measured in control and DEDC-exposed rat nerves. (A) MDA levels were determined in posterior tibial nerves by HPLC MDA values are means ± SEM for each time point (the number of nerves analyzed were n=26 for the control zero time point, n=8 for the 2 and 4-week time points and n= 12 for the 8-week time point) two separate analyses were performed per nerve and both were assayed in duplicate by HPLC, giving 4 values per nerve.. (B) F₂-Isoprostane levels in sciatic nerve were determined by the modified method of Folch and quantified by GC/MS. Isoprostane values are means ± SEM for each time point (n=4, except for DEDC 8-week n=5). *p < 0.05 and+p < 0.01, One-way ANOVA and Dunnett Multiple Comparisons Test as compared to zero time point.

Figure 3

Relative amounts of HNE protein adducts in sciatic nerve proteins from DEDC-exposed groups and their corresponding controls. Total sciatic nerve proteins (0.2 μg) from each control and DEDC treatment group (n=4, except for 8-week time point, control (n=5) and DEDC-exposed (n=6)) were applied in triplicate to a PVDF membrane using a slot blot apparatus and evaluated for HNE adducts by immunoassay (see methods section) using a rabbit anti-HNE antibody. Data are expressed as means ± SEM of four membranes for each animal. Values were normalized so that optical density of controls was 1.0. * p < 0.05 by unpaired one-tailed t test as compared to the corresponding control group.

Figure 4

IgG and IgG2a levels in sciatic nerve proteins of control and DEDC-exposed groups. Total sciatic nerve protein (0.2 μg) from each control and DEDC-exposed rat were evaluated for IgG and IgG2a levels using a slot blot apparatus and immunoassay (see methods for details). (n=4, except for 8-week time point, control (n=5) and DEDC-exposed (n=6)) Values are means ± SEM. of four membranes with individual animals run in duplicate on each membrane. Values were normalized so that optical density of controls was 1.0. *p < 0.05, unpaired two-tailed t test as compared to the corresponding exposure duration controls.

Figure 5

Immunolocalization of activated macrophages in representative cross-sections of sciatic nerves from an 8-week control rat (A) and an 8-week DEDC exposed rat (B) probed with mouse-anti-rat CD68 and counterstained with hematoxylin. Some activated macrophages are identified with arrows. (C) A serial cross-section to that shown in B from the 8-week DEDC exposed rat that was processed identically to the sections in A and B, but without mouse-anti-rat CD68 primary antibody.

Figure 6

Morphology of sciatic nerve cross-sections stained with toluidine blue. (A) Sciatic nerve from a control rat showing axons surrounded by normal compact myelin. (B) Cross section of a nerve obtained from an 8-week DEDC-exposed rat showing examples of thinly myelinated (arrows) and demyelinated axons (concave arrow head) and intramyelinic edema (arrow heads).

Figure 7

Electron micrographs of sciatic nerve cross-sections from control and DEDC-treated rats (black bars = 2μm). (A) Myelinated axon (a) in a control animal surrounded by normal compact myelin. (B-D) Sections from a rat exposed to DEDC for 8-weeks showing lesions that were quantified in Table 1. (B) A myelinated axon (a) surrounded by abnormally thin compact myelin. (C) Myelinated axon (a) with fluid separating the inner lamellae of myelin (*) (intramyelinic edema). (D) A demyelinated axon (a) from an 8-week DEDC exposed rat is surrounded by a Schwann cell containing a non-membrane vacuole. Two nucleated cells possessing basal lamina consistent with Schwann cells are also present (s).