Dietary Copper Enhances the Peripheral Myelinopathy Produced by Oral P
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《毒物学科学杂志》
Department of Pathology, Vanderbilt University Medical Center, Nashville, Tennessee 37232–2561
ABSTRACT
The neurotoxic hazard of a dithiocarbamate is influenced by route of exposure and acid stability of the dithiocarbamate. As an example, oral administration of the acid labile dithiocarbamate N,N-diethyldithiocarbamate (DEDC) causes a central-peripheral axonopathy thought to result from acid-promoted decomposition to CS2 in the stomach. In contrast, parenteral administration of DEDC, which bypasses the acidic environment of the stomach, causes a primary demyelination that is thought to be mediated through the intact parent dithiocarbamate. The relative acid stability of pyrrolidine dithiocarbamate (PDTC) suggests that a significant portion of a dose can be absorbed intact following oral exposure with the potential to produce a primary myelin injury. The present study was performed to characterize the neurotoxicity of PDTC and evaluate the possible role of copper in dithiocarbamate-mediated demyelination. Male Sprague Dawley rats were administered PDTC in drinking water and given either a normal- or high-copper diet for 18, 47, or 58 weeks. Examination of peripheral nerve by light microscopy and electron microscopy at the end of exposures revealed primary myelin lesions and axonal degeneration in the PDTC groups, with a significant increase in the severity of several lesions observed for the PDTC, high-copper group relative to the PDTC normal-copper diet. ICP-AES metal analysis determined that the PDTC groups had significantly increased brain copper, and at 58 weeks a significant increase in copper was seen in the sciatic nerve of PDTC high-copper animals relative to PDTC normal-copper diet animals. Although RP-HPLC analysis could not detect globin alkylaminocarbonyl cysteine modifications analogous to those seen with parenteral DEDC, LC/MS/MS identified (pyrrolidin-1-yl carbonyl)cysteine adducts on PDTC-exposed rat globin. These findings are consistent with previous studies supporting the ability of acid-stable dithiocarbamates to mediate myelin injury following oral exposure. The greater severity of lesions associated with dietary copper supplementation and elevated copper levels in nerve also suggests that perturbation of copper homeostasis may contribute to the development of myelin lesions.
Key Words: copper; sodium pyrrolidine dithiocarbamate; dithiocarbamate; demyelination; myelin; peripheral neuropathy.
INTRODUCTION
Human exposures to dithiocarbamates and their disulfides results from their applications in agriculture, manufacturing, and medicine (Eneanya et al., 1981; Haley, 1979; WHO, 1988). Adverse effects from dithiocarbamates were recognized more than half a century ago, when observations were made that disulfiram, (bis(diethylthiocarbamoyl)disulfide), could cause a dose-dependent distal, sensorimotor neuropathy when used to treat alcoholism. Dithiocarbamates also have widespread uses as pesticides, with the potential for human exposure from food crop residues, groundwater contamination, and occupational exposure during application (Alexeeff et al., 1994; Kreutzer et al., 1994; Vettorazzi et al., 1995). In addition, dithiocarbamates have been used to treat nickel intoxication (Brewer, 1993; Jones and Jones, 1984), and other medical uses for PDTC and DEDC are under investigation, including applications in clinical oncology (Bach et al., 2000; Chinery et al., 1997; DeWoskin and Riviere, 1991).
Previous studies have also provided evidence for the development of neurotoxicity due to dithiocarbamate exposure in humans and animals (Forns et al., 1994; Frisoni and Di Monda, 1989; Rasul and Howell, 1973). Both the chemical structure and route of exposure influence the disposition and type of neurotoxicity produced. For example, oral administration of acid-labile N,N-diethyldithiocarbamate (DEDC) produces biologically significant amounts of carbon disulfide (CS2) through acid-promoted decomposition in the stomach and results in CS2-mediated protein cross-linking and the development of a neurofilamentous axonopathy identical to that produced by CS2 (Johnson et al., 1998). In contrast, parenteral administration of DEDC or oral administration of the more acid-stable disulfide dimer of DEDC, disulfiram, is characterized by the generation of S-(diethylaminocarbonyl)cysteine adducts in the absence of CS2-mediated protein cross-linking and the development of segmental demyelination (Tonkin et al., 2000, 2003). These observations have led to the interpretation that the intact dithiocarbamate is the proximate toxic species for the myelin lesions. In the case of disulfiram, this acid-stable parent compound can be absorbed intact following oral administration and then reduced to DEDC in vivo in a neutral environment, reproducing the exposure produced by parenteral administration of DEDC.
Copper has been established to be or is currently being considered as a contributing agent in the development of a number of neurodegeneratie diseases including Alzheimer's disease, amylotrophic lateral sclerosis, Wilson's disease, Menke's disease, and prion diseases (Multhaup et al., 2002; Rotilio et al., 2002; Strusak et al., 2001). Accumulation of copper in the nervous system has also been proposed as a contributing mechanism for the neurotoxicity of dithiocarbamates, and previous experiments have supported a role for this mechanism. In vitro studies using primary rat astrocytes or thymocytes have associated intracellular transport of copper and enhanced oxidative stress with increased cytotoxicity (Chen et al., 2000; Orrenius et al., 1996; Wilson and Trombetta, 1999). Cytotoxicity in those cell lines was decreased by preventing accumulation of intracellular copper through incubation of the cells in copper-free media or coadministration of a hydrophilic, non-cell-permeable copper chelator. Previous in vivo studies have also demonstrated increased copper levels and lipid peroxidation in sciatic nerve and brain resulting from intraperitoneal administration of DEDC (Tonkin et al., 2004) or oral disulfiram (Delmaestro and Trombetta, 1995) and oral administration of PDTC (Calviello et al., 2005). In the present investigation, PDTC, a more acid-stable cyclic dithiocarbamate relative to DEDC, was administered orally to rats to determine whether it would produce myelin-associated lesions similar to those observed for parenteral DEDC and oral disulfiram, or alternatively, the CS2-mediated axonopathy previously observed following oral administration of DEDC. Rats were also either fed a diet containing 13 ppm or 200 ppm copper to determine if increased dietary copper would enhance copper accumulation or the neurotoxicity of PDTC. The water solubility and tolerance of rats to the dosing of PDTC in drinking water made it possible to produce a chronic exposure, presumably maximizing accumulation of copper in the nervous system, and to assess the potential of dithiocarbamates to produce a cumulative effect. Following exposures, body weights and hindlimb grip strengths were compared, and lesions in peripheral nerve were assessed using light and electron microscopy. Brain, liver, and peripheral nerve metals, including copper, were measured by inductively coupled plasma-atomic emission spectroscopy (ICP-AES), and serum copper was measured by RP-HPLC. Analysis of globin to evaluate in vivo release of CS2 and the potential of PDTC to produce S-(pyrrolidin-1-ylcarbonyl)cysteine (PDC-Cys) adducts analogous to those generated by DEDC and disulfiram was performed by RP-HPLC and LC/MS/MS.
MATERIALS AND METHODS
Chemicals.
Glutaraldehyde was obtained from Electron Microscopy Sciences (Ft. Washington, PA). Laboratory rodent diet (Purina Laboratory Diet, 5001) containing 13 ppm copper and the same diet supplemented to 200 ppm copper using copper sulfate were obtained from Purina Mills (Richmond, IN). CS2 was obtained from EM Sciences (Gibbstown, NJ), and pyrrolidine was purchased from Alfa Aesar (Ward Hill, MA).
Chemical Syntheses
Pyrrolidine dithiocarbamate.
Freshly distilled pyrrolidine (14.25 g, 0.2 mole) was taken in a mixure of ethanol (125 ml) and water (75 ml), and stirred in an ice bath. Carbon disulfide (15 ml, 0.25 mole) was added, followed by 10 N NaOH (20 ml). After 1 h the precipitated solid was washed with absolute ethanol and dried; 30 g (88%). The identity and purity (>99%) of pyrrolidine dithiocarbamate was verified by NMR spectroscopy and by UV Spectrophotometry.
S-(Piperidin-1-ylcarbonyl)cysteine.
S-(Piperidin-1-ylcarbonyl)cysteine (Pip-cys) was synthesized as previously described (Zimmerman et al., 2004). TEAB-ACN buffer was prepared from 1 M triethylammonium bicarbonate (pH 8.0, 2 ml), triethylamine (0.5 ml), water (50 ml), and acetonitrile (50 ml).
Animals and exposures.
All exposures were conducted in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee. Male Sprague-Dawley rats obtained from Harlan Bioproducts (Indianapolis, IN) were given food and water ad libitum and caged in a room on a diurnal light cycle. Body weights were determined prior to the start of the study and then weekly during the course of the experiment. Four exposure groups were used: two PDTC-exposed groups receiving 8 mM PDTC in deionized water and two control groups given tap water. One control group (n = 8) and one PDTC group (n = 8) were given Purina rodent diet 5001 containing 13 ppm copper (standard rat maintenance diet fed at Vanderbilt University), and the remaining control group (n = 8) and PDTC group (n = 8) were given Purina rodent lab diet 5001 containing 200 ppm copper. The 200 ppm copper diet was chosen for copper loading after literature review indicated no significant clinical, chemical, histopathological, and hematological toxicity at this level when chronically administered (Cristofori et al., 1992; Everling et al., 1990; Fuentealba et al., 2000; Milanino et al., 2000), while still affording a 15-fold increase in dietary copper. The average starting weight of the 13 ppm copper diet animals was 291 ± 3 g, and the average starting weight of the 200 ppm copper diet animals was 401 ± 6 g. PDTC was delivered as an aqueous solution of NaPDTC in deionized water, the concentration of which was determined from the UV absorbance at 277 nm ( = 13,540 M–1 cm–1). The dosing of PDTC in the drinking water of both normal copper and high copper treatment groups was accomplished gradually (to avoid rejection of the solution) by increasing the concentration from 4 mM to 8 mM over a period of 21 days. This was the maximum oral tolerated dose. Water intake between groups was compared at the beginning of week 16 of the study and was quantified by measurement of water intake per cage (four animals per cage) over a 10-day period. Four of the 13 ppm copper PDTC and 13 ppm copper control rats were analyzed first at 18 weeks to look for differences in tissue copper levels and to detect early peripheral nerve lesions, and the remaining 13 ppm copper animals were taken out to 58 weeks. Three of the 200 ppm copper PDTC and 200 ppm copper control rats were analyzed at 47 weeks when three animals in the 200 ppm copper PDTC group had to be removed from the study due to significant body weight loss and paresis of their hindlimbs, and at that time, one other 200 ppm copper PDTC animal with the lowest grip strength was sacrificed along with matched controls. The remaining 200 ppm copper PDTC and control animals were continued to 58 weeks when significant paresis and weight loss was evident. At each time point examined, neuromuscular function was assessed by measuring hindlimb grip strength using a digital force gauge (Meyer et al., 1979), and tissues were collected for analyses (see below).
Blood collection.
After induction of deep anesthesia, whole blood (3–4 ml) was collected from the left ventricle prior to perfusion or tissue collection. A volume of 0.6–1.0 ml whole blood was placed into a heparinized Eppendorf tube for globin isolation and subsequent analysis by HPLC or LC/MS/MS; and 2–3 ml was allowed to clot in serum separator tubes, and serum was collected after centrifugation for copper analysis by RP-HPLC (see below). Heparanized blood was separated into plasma and hemolysate as previously described (Erve et al., 2000), and 100 μl of 1 M ascorbic acid was added to 1 ml of hemolysate. The resulting solution was added dropwise to 7 ml of 2.5% oxalic acid in acetone. Globin was allowed to precipitate on ice for 15 min, and then centrifuged at 10,000 x g for 10 min at 4°C, washed in 5 ml ice cold acetone, and centrifuged at 10,000 x g for 10 min. The resulting pellet, containing crude globin, was dried under a nitrogen stream and stored at –80°C. Dried globin was solubilized with 0.1% trifluroacetic acid (TFA) to produce a solution for HPLC analysis.
Globin chains were separated by RP-HPLC on a Phenomenex Jupiter 5-μm, 300 column (150 x 460 mm) using a Waters 2690 liquid chromatograph after adjusting sample concentration to a UV absorption of 1 at 280 nm. Globins were separated using a linear gradient from 56:44% (Solvent A:Solvent B) to 30:70% (A:B) over 30 min, followed by a linear gradient to 100% solvent B (1 min.) holding for 5 min, reequilibrating to 56:44 (A:B) over 10 min, and holding for 10 min. Solvent A was 20:80:0.1 acetonitrile:water:TFA, and solvent B was 60:40:0.08 acetonitrile:water:TFA. The elution of globin peaks was monitored by their UV absorption at 220 nm using a Waters 996 photodiode array detector.
Preparation of nerve tissue and morphological assessments.
Animals examined at 18 weeks were perfused through the left ventricle of the heart under deep anesthesia (100 mg/kg ketamine HCL plus 15 mg/kg xylazine) with a solution of 0.8% NaCl, 0.025% KCl, 0.05% NaHCO3, in 0.01 M phosphate buffer followed by 4% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. Animals examined at 47 and 58 week timepoints were exsanguinated under deep anesthesia and not perfused. After exsanguination or perfusion, liver, brain, and sciatic nerves were dissected out and immersed in 4% glutaraldeyde in 0.1 M phosphate buffer overnight and then transferred to 0.1 M phosphate buffer (pH 7.4). Sciatic nerve sections were post-fixed with osmium and embedded in Epon; thick sections were cut and stained with toluidine blue. Thick (1 μm) sections of peripheral nerve tissue were evaluated by light microscopy on an Olympus BX41 microscope equipped with an Optronics Microfire digital camera. Four different fields at 40x (total area = 0.26 mm2) were photographed from one section of each sciatic nerve, and the total number of lesions counted by one observer (HLV). The lesions quantified were: degenerated axons, axons with thin myelin (myelin sheath with an axon/fiber diameter ratio (g ratio) of >0.7 (King, 1999)), intramyelinic edema, and demyelinated axons. Thin (70 nm) sections were prepared from sciatic nerves and evaluated using a Phillips CM-12 electron microscope, 120 keV with a high resolution CCD camera system.
Analysis of tissue metal levels.
Tissue sections of brain, liver, and sciatic nerve were analyzed by inductively coupled plasma-atomic emission spectroscopy (ICP-AES) at the Diagnostic Center for Population and Animal Health at Michigan State University (East Lansing, MI). Elements determined in this analysis were copper, calcium, iron, and zinc. Individual liver and brain samples were analyzed in duplicate for mineral content, and the results reported as one value per individual. One sciatic nerve was taken from each animal and pooled with other members of the same treatment group to provide adequate tissue for ICP-AES analysis, and this resulted in one analysis injected twice per group, thus generating two values per group. Brain tissue samples for ICP-AES were approximately 2-mm midsagittal slices; liver sections were approximately 1-cm3 pieces of tissue; sciatic nerve sections were approximately 15- to 20-mm lengths of a single nerve.
Analysis of serum copper levels.
Serum (100 μl) was mixed with 6 M HCl (20 μl) at ambient temperature for 10 min before cooling in ice and centrifugation (13, 000 x g) for 16 min. An aliquot (80 μl) of the supernatant was neutralized with 20 μl 1 M K2HPO4 and 30 μl 4 M NH4OH, and treated with 40 μl of 20 mM bis-hydroxyethyl dithiocarbamate (HEDC). After 10 min the copper complex was extracted with ethyl acetate (2 x 400 μl), solvent evaporated under a stream of nitrogen, and residue dissolved in 50 μl of 60:40 10 mM ammonium acetate-acetonitrile. The solvent mixture of 60:40 water:acetonitrile was used for analyzing 5 μl of the reconstituted solution by HPLC on an Alltex Alltima HP C18 reversed-phase column (2.1 x 150 mm; 5 μm). A photo-diode array detector in the wavelength range 360 to 460 nm was used to monitor the elution, and absorbance at 430 nm was used for measurements. A standard curve using known concentrations of Cu-HEDC from 0 to 250 μM was generated.
LC/MS/MS analysis of globins.
Globin samples (5–10 mg) were hydrolyzed with 6 M HCl for 4 h, and excess acid was removed. Internal standard (5 μl of 100 μM Pip-cys) was added to the residue, which was dissolved in TEAB-ACN buffer (200 μl), and triethylamine (10 μl), and 3-pyridylisothiocyanate (40 μl of a 50 μM solution in ACN) were added to this. The mixture was agitated in a water bath at 37°C for 1 h and evaporated. The residue was reconstituted with 10 mM HCl (100 μl), and a 10 μl aliquot was analyzed by LC/MS/MS. An Exterra MS C18 column (2.1 x 100 mm; 3.5 μm) running at 0.2 ml/min was used. A gradient elution from 100% A (10% methanol in 5 mM formic acid) to 60% B (100% methanol in 5 mM formic acid) over 20 min was used to elute the analyte and the standard. Selected reaction monitoring (SRM) using the daughter ion m/z 137 generated from the molecular ions 355 (PDC-Cys adduct) and 369 (Pip-Cys adduct) was used for measurements. Known amounts of PDC-Cys and Pip-Cys were taken, dried, derivatized, and analyzed by SRM. Mole ratios of the two were plotted against area ratios to generate a standard curve. Amounts of PDC-Cys were estimated using the calculated response factor.
Statistical analysis.
One-way analysis of variance (ANOVA) with Tukey-Kramer's Multiple Comparisons Test and the one-tailed t-test were performed using GraphPad Software, Inc. Lesion counts were square root transformed to normalize their distribution and equalize variances prior to statistical comparisons by ANOVA and Tukey-Kramer's Multiple Comparisons Test. For two group comparisons where equal variances were not obtained as determined by the F test, the t-test corrected for unequal variance was used. Statistical significance was taken to be p < 0.05 unless otherwise noted.
RESULTS
Body Weights and Dosage Levels
Administration of PDTC resulted in significantly reduced body weights relative to controls at each time point (Table 1). Since at the 58-week time point, the body weights of same strain male animals were expected to be similar in the absence of dietary copper manipulations or PDTC administration, all groups were compared regardless of average starting weights. No significant effect on body weight from increased dietary copper alone was apparent.
The average dose of PDTC taken in the water was determined by measuring water volume intake every 2 days per group over 10 days beginning at week 16. A value of 10.5 ml of 8 mM PDTC/100 g BW value was obtained on rats given PDTC eating a 13 ppm copper diet, and an average of 11.0 ml of 8 mM PDTC/100 g BW was obtained on rats given PDTC eating a 200 ppm copper diet. This was calculated to be 0.85 mmol/kg/day for PDTC-treated, 13 ppm copper diet animals and 0.88 mmol/kg/day for PDTC-treated, 200 ppm copper diet animals.
Analysis of Tissue Copper Levels
Tissue copper levels for each time point are presented in Figure 1. Brain copper levels in all PDTC-exposed groups were significantly elevated over same-diet controls. As the study progressed from 18 to 58 weeks, brain copper levels increased in both the control and PDTC-treated rats, with those in the combined PDTC and high-copper diet containing the greatest levels. Similar to brain, sciatic nerve copper was also the greatest in the groups on the combined PDTC high-copper diet, and sciatic nerve copper appeared to accumulate as a function of age in controls, with more accumulation occurring in the high copper diet controls.
In contrast, liver copper was only significantly increased in PDTC-exposed animals that also received the 200 ppm copper diet at the 47 and 58 week time points.
The only significant difference noted for serum copper levels that appeared to be dithiocarbamate-related was an increase in the 47-week PDTC 200 ppm copper group relative to the 47-week control 200 ppm copper group.
Analysis of Tissue Calcium, Iron, and Zinc
Although there were some significant differences between treatment groups at all three timepoints, there were no trends with any of the three minerals consistent with compound-related changes. These results are presented as supplementary data.
Globin Analyses
Additional late-eluting peaks resulting from covalent modification of the globin 3 chain analogous to those produced by parenteral DEDC administration were not detected in any of the PDTC-dosed rats at either 18, 47, or 58 weeks.
The greater sensitivity afforded by LC/MS/MS analysis made it possible to demonstrate the presence of PDC-Cys adducts on globin (Erve et al., 2000; Tonkin et al., 2000, 2003). At 58 weeks, the level of PDC-Cys on globin for 200 ppm PDTC-exposed animals was significantly greater than for PDTC, 13 ppm copper diet animals and both control groups (Table 2).
Neuromuscular Function and Peripheral Nerve Morphology
Significant decreases in grip strength were observed at each timepoint for PDTC-dosed rats relative to their respective controls (Table 3). In addition, administration of the high-copper diet with PDTC produced a significant decrease in grip strength relative to PDTC with the 13 ppm copper diet. No difference in grip strength resulted from 200 ppm copper diet alone relative to controls on the 13 ppm copper diet.
Representative sections of sciatic nerve obtained from controls and animals exposed to PDTC demonstrating the lesions quantified in Table 4 are shown at the light microscope level in Figure 2 and electron microscope level in Figure 3. Peripheral nerve lesions were observed both in animals exposed to PDTC alone and in animals exposed to PDTC in combination with a high-copper diet that were not present in control animals on either diet. A significant increase in axons exhibiting thin myelin and undergoing degeneration was observed in the 18-week, PDTC group. At 47 weeks, two animals in the PDTC 200 ppm copper group had become very weak in their hind legs and had lost a substantial amount of body weight. They were removed from the study along with one other PDTC, 200 ppm copper rat with the next lowest grip strength along with three 200 ppm copper controls, and their tissues were collected. The two most affected PDTC, 200 ppm animals exhibited much higher numbers of degenerated axons, thinly myelinated axons, intramyelinic edema, and demyelinated axons than the other affected PDTC, 200 ppm animal, and the number of demyelinated and degenerated axons in the PDTC 200 ppm copper group was significantly elevated over the 200 ppm controls. At 58 weeks, the remaining PDTC 200 ppm copper-exposed animals were clinically weak and had significant weight loss. Also at 58 weeks, a significant increase in thinly myelinated axons, intramyelinic edema, and degenerated axons was seen in the PDTC 200 ppm copper animals as compared to the PDTC 13 ppm copper animals, the 200 ppm controls, and the 13 ppm controls. Also a significant increase in demyelinated axons was seen in the PDTC 200 ppm copper animals as compared to the 200 ppm and 13 ppm controls. Axonal regeneration and remyelination was evidenced by the presence of axonal sprouts and onion bulbs, respectively.
DISCUSSION
Numerous biological effects have been reported for pyrrolidine dithiocarbamate in vitro and in vivo including pro-oxidant, antioxidant, chelation, antiprotozoal, antiviral, and antineoplastic effects, but relatively little information is available regarding its potential neurotoxicity in vivo.
Due to the pyrrolidine ring structure, PDTC is substantially more resistant to acid-promoted decomposition to parent amine and CS2 than noncyclic aliphatic dialkyl dithiocarbamates, e.g., DEDC, and this has been the principal reason for using it instead of DEDC within biological systems. The relative acid stability of PDTC suggests that a greater percentage of an oral dose would be absorbed intact as parent dithiocarbamate relative to DEDC. Consistent with this interpretation, there was no evidence for CS2-mediated intramolecular protein cross-linking of the -globin chain observed by HPLC in this study. Thus, if parent dithiocarbamate is the proximate toxic species for the myelin lesions as previously proposed, PDTC would be predicted to be a Schwann cell toxicant similar to disulfiram and parenteral DEDC. This was indeed the case, and the structural changes produced in peripheral nerve by oral PDTC corresponded to a chronic demyelinating injury, evidenced by naked axons, onion bulb formation, intramyelinic edema, and thinly myelinated axons. Axonal degeneration was also observed. In many cases this appeared to be a secondary axonal degeneration subsequent to Schwann cell injury, but the observation of axonal degeneration at the earliest time point suggests that there may be some primary axonal involvement, although thinned myelin was also a significant finding at this time point.
Although carbamylation of proteins by dithiocarbamates does not appear to underlie the demyelinating injury produced by dithiocarbamates (Tonkin et al., 2003), it has been used as an internal dosimeter and for monitoring the distribution of dithiocarbamates in vivo (Erve et al., 2000; Tonkin et al., 2004; Zimmerman et al., 2004). Additionally, cysteine carbamylation is thought to be responsible for inhibition of aldehyde dehydrogenase, which is the basis for using disulfiram in alcohol aversion therapy (Shen et al., 2001; Zimmerman et al., 2004). In the current study, analysis of globin samples by LC/MS/MS revealed that PDTC also carbamylates cysteine residues on globin, but to a much lower extent than DEDC or disulfiram, as evidenced by HPLC. For PDTC there were no readily identifiable new HPLC peaks in globin preparations, whereas neurotoxic doses of parenteral DEDC and oral disulfiram (Tonkin et al., 2000, 2003) have been observed to modify up to 13 to 24% of the chains of globin. At least two interpretations for this finding are possible. Modification of Cys-125 on the 3 chain of globin by PDTC may not alter its retention time to the same extent as DEDC, or more likely, PDTC is not metabolized to a reactive sulfoxide or sulfone intermediate to the same extent as DEDC.
Although zinc, iron, and calcium were also measured in tissues, no trends were noted like those seen with copper. This is consistent with a previous report (Calviello et al., 2005), where an elevation in tibial nerve copper occurred without changes in zinc or iron following oral administration of PDTC. This may be due to the formation of unstable PDTC chelates with these other ions, rendering them unable to enter lipid-containing tissues. Interestingly, the 58-week PDTC 200 ppm Cu group had significantly greater severity scores for most categories compared to the other 58-week groups, including the PDTC 13 ppm copper group. These data, together with the elevated copper levels present in the sciatic nerves of the PDTC high-copper diet animals, suggest that disruption of copper homeostasis by PDTC may contribute to the development of lesions in peripheral nerve and that PDTC is able to do this through the formation of a nonionic chelate with Cu2+ which can cross the blood–brain barrier and accumulate in lipid-containing tissues. Additionally, oral administration of PDTC increased the level of copper in brain and had negative effects on weight gain and hind limb grip strengths. Similar to the situation observed for peripheral nerve lesions, dietary copper supplementation also enhanced the effects of PDTC for these three parameters. In contrast, elevations in liver and serum copper were only observed in the POTC groups fed the high-copper diet. Copper levels in sciatic nerve were also clearly elevated in the groups administered both PDTC and the high-copper diet, but the effect of PDTC alone was less clear. Although there appeared to be a trend for PDTC to increase copper in sciatic nerve at the early time point, this trend was not verified at 58 weeks. Since elevations of brain copper produced by PDTC with the normal copper diet paralleled previous results reported for DEDC, and DEDC is able to increase sciatic nerve copper without increased dietary copper, the values obtained for sciatic nerve copper levels in the PDTC normal copper diet animals at 58 weeks may be anomalous. Unfortunately the quantity of sample needed for ICP-AES required pooling of the nerves, decreasing the n and preventing reanalysis of the samples. Therefore, further investigation will be required to unequivocally resolve this question.
The findings of the present study are consonant with previous studies in which routes of exposure and chemical properties that favored absorption of intact dithiocarbamate with copper accumulation in the nervous system were associated with myelinopathy. Additional evidence was obtained that the level of copper accumulated by a dithiocarbamate may be positively correlated to the severity of injury within peripheral nerve. Considering the tight regulation of copper within biological systems and the extremely limited amount of free copper (Rae et al., 1999), the magnitude of changes in copper levels observed here could have substantial biological effects if only a very small fraction of the increased copper existed as free copper. Indeed, previous studies have suggested a role for copper-promoted oxidative stress in dithiocarbamate-mediated neurotoxicity based upon associated elevations in markers for lipid peroxidation (Delmaestro and Trombetta, 1995; Tonkin et al., 2004). It should be noted, though, that these analyses were performed at advanced endpoints and, at least in the case of peripheral nerve, were accompanied by substantial injury. This raises the possibility that the elevation of lipid peroxidation products observed might not be a specific finding for the dithiocarbamate, but possibly a more general consequence of the ongoing demyelinating process accompanied by the influx of active macrophages. In contrast, evidence has also been provided that deficiencies of copper can result in myelinopathies (Dake and Amemiya, 1991; Zimmerman et al., 1976), and the potential for dithiocarbamates to sequester copper and limit its availability as a cofactor for certain enzymes (Heikkila et al., 1976), e.g., copper zinc superoxide dismutase and dopamine hydroxylase, presents an alternative hypothesis. But the more severe lesions obtained for the high-copper diet animals appears contradictory to this interpretation. To ascertain the role of dithiocarbamate-mediated alteration of copper homeostasis in peripheral nerve injury, further investigation is required. Studies directed at determining the chemical species responsible for copper elevation in nerve, defining the distribution of elevated copper in nerve and delineating the temporal relationship of oxidative injury to the onset of structural changes will provide useful information toward this goal. The significance of defining the role of copper accumulation in dithiocarbamate neurotoxicity could be realized through facilitating the development of structure–activity relationships useful for defining the relative risks associated with various dithiocarbamates and designing safer dithiocarbamates.
SUPPLEMENTARY DATA
Zinc, iron and calcium levels measured by ICP-AES for liver, brain and sciatic nerve tissue are provided as supplementary data in a word document named Zn, Fe, Ca suppdata.doc. Supplementary data are available online at www.toxsci.oxfordjournals.org.
ACKNOWLEDGMENTS
The authors would like to thank Dr. Doyle Graham for his consultation and mentoring of peripheral nerve pathology. NMR analysis of pyrrolidine dithiocarbamate was performed by Dr. Lisa Zimmerman. LC/MS/MS analysis was performed with the assistance of Lisa Manier of the Mass Spectrometry Research Center at Vanderbilt University. Experiments were performed in part through the use of the VUMC Research EM Resource (sponsored by NIH Grants DK20539 and DK58404). This work was supported by NIEHS Grant ES06387 and by the Center of Molecular Toxicology Grant P30 ES00267.
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ABSTRACT
The neurotoxic hazard of a dithiocarbamate is influenced by route of exposure and acid stability of the dithiocarbamate. As an example, oral administration of the acid labile dithiocarbamate N,N-diethyldithiocarbamate (DEDC) causes a central-peripheral axonopathy thought to result from acid-promoted decomposition to CS2 in the stomach. In contrast, parenteral administration of DEDC, which bypasses the acidic environment of the stomach, causes a primary demyelination that is thought to be mediated through the intact parent dithiocarbamate. The relative acid stability of pyrrolidine dithiocarbamate (PDTC) suggests that a significant portion of a dose can be absorbed intact following oral exposure with the potential to produce a primary myelin injury. The present study was performed to characterize the neurotoxicity of PDTC and evaluate the possible role of copper in dithiocarbamate-mediated demyelination. Male Sprague Dawley rats were administered PDTC in drinking water and given either a normal- or high-copper diet for 18, 47, or 58 weeks. Examination of peripheral nerve by light microscopy and electron microscopy at the end of exposures revealed primary myelin lesions and axonal degeneration in the PDTC groups, with a significant increase in the severity of several lesions observed for the PDTC, high-copper group relative to the PDTC normal-copper diet. ICP-AES metal analysis determined that the PDTC groups had significantly increased brain copper, and at 58 weeks a significant increase in copper was seen in the sciatic nerve of PDTC high-copper animals relative to PDTC normal-copper diet animals. Although RP-HPLC analysis could not detect globin alkylaminocarbonyl cysteine modifications analogous to those seen with parenteral DEDC, LC/MS/MS identified (pyrrolidin-1-yl carbonyl)cysteine adducts on PDTC-exposed rat globin. These findings are consistent with previous studies supporting the ability of acid-stable dithiocarbamates to mediate myelin injury following oral exposure. The greater severity of lesions associated with dietary copper supplementation and elevated copper levels in nerve also suggests that perturbation of copper homeostasis may contribute to the development of myelin lesions.
Key Words: copper; sodium pyrrolidine dithiocarbamate; dithiocarbamate; demyelination; myelin; peripheral neuropathy.
INTRODUCTION
Human exposures to dithiocarbamates and their disulfides results from their applications in agriculture, manufacturing, and medicine (Eneanya et al., 1981; Haley, 1979; WHO, 1988). Adverse effects from dithiocarbamates were recognized more than half a century ago, when observations were made that disulfiram, (bis(diethylthiocarbamoyl)disulfide), could cause a dose-dependent distal, sensorimotor neuropathy when used to treat alcoholism. Dithiocarbamates also have widespread uses as pesticides, with the potential for human exposure from food crop residues, groundwater contamination, and occupational exposure during application (Alexeeff et al., 1994; Kreutzer et al., 1994; Vettorazzi et al., 1995). In addition, dithiocarbamates have been used to treat nickel intoxication (Brewer, 1993; Jones and Jones, 1984), and other medical uses for PDTC and DEDC are under investigation, including applications in clinical oncology (Bach et al., 2000; Chinery et al., 1997; DeWoskin and Riviere, 1991).
Previous studies have also provided evidence for the development of neurotoxicity due to dithiocarbamate exposure in humans and animals (Forns et al., 1994; Frisoni and Di Monda, 1989; Rasul and Howell, 1973). Both the chemical structure and route of exposure influence the disposition and type of neurotoxicity produced. For example, oral administration of acid-labile N,N-diethyldithiocarbamate (DEDC) produces biologically significant amounts of carbon disulfide (CS2) through acid-promoted decomposition in the stomach and results in CS2-mediated protein cross-linking and the development of a neurofilamentous axonopathy identical to that produced by CS2 (Johnson et al., 1998). In contrast, parenteral administration of DEDC or oral administration of the more acid-stable disulfide dimer of DEDC, disulfiram, is characterized by the generation of S-(diethylaminocarbonyl)cysteine adducts in the absence of CS2-mediated protein cross-linking and the development of segmental demyelination (Tonkin et al., 2000, 2003). These observations have led to the interpretation that the intact dithiocarbamate is the proximate toxic species for the myelin lesions. In the case of disulfiram, this acid-stable parent compound can be absorbed intact following oral administration and then reduced to DEDC in vivo in a neutral environment, reproducing the exposure produced by parenteral administration of DEDC.
Copper has been established to be or is currently being considered as a contributing agent in the development of a number of neurodegeneratie diseases including Alzheimer's disease, amylotrophic lateral sclerosis, Wilson's disease, Menke's disease, and prion diseases (Multhaup et al., 2002; Rotilio et al., 2002; Strusak et al., 2001). Accumulation of copper in the nervous system has also been proposed as a contributing mechanism for the neurotoxicity of dithiocarbamates, and previous experiments have supported a role for this mechanism. In vitro studies using primary rat astrocytes or thymocytes have associated intracellular transport of copper and enhanced oxidative stress with increased cytotoxicity (Chen et al., 2000; Orrenius et al., 1996; Wilson and Trombetta, 1999). Cytotoxicity in those cell lines was decreased by preventing accumulation of intracellular copper through incubation of the cells in copper-free media or coadministration of a hydrophilic, non-cell-permeable copper chelator. Previous in vivo studies have also demonstrated increased copper levels and lipid peroxidation in sciatic nerve and brain resulting from intraperitoneal administration of DEDC (Tonkin et al., 2004) or oral disulfiram (Delmaestro and Trombetta, 1995) and oral administration of PDTC (Calviello et al., 2005). In the present investigation, PDTC, a more acid-stable cyclic dithiocarbamate relative to DEDC, was administered orally to rats to determine whether it would produce myelin-associated lesions similar to those observed for parenteral DEDC and oral disulfiram, or alternatively, the CS2-mediated axonopathy previously observed following oral administration of DEDC. Rats were also either fed a diet containing 13 ppm or 200 ppm copper to determine if increased dietary copper would enhance copper accumulation or the neurotoxicity of PDTC. The water solubility and tolerance of rats to the dosing of PDTC in drinking water made it possible to produce a chronic exposure, presumably maximizing accumulation of copper in the nervous system, and to assess the potential of dithiocarbamates to produce a cumulative effect. Following exposures, body weights and hindlimb grip strengths were compared, and lesions in peripheral nerve were assessed using light and electron microscopy. Brain, liver, and peripheral nerve metals, including copper, were measured by inductively coupled plasma-atomic emission spectroscopy (ICP-AES), and serum copper was measured by RP-HPLC. Analysis of globin to evaluate in vivo release of CS2 and the potential of PDTC to produce S-(pyrrolidin-1-ylcarbonyl)cysteine (PDC-Cys) adducts analogous to those generated by DEDC and disulfiram was performed by RP-HPLC and LC/MS/MS.
MATERIALS AND METHODS
Chemicals.
Glutaraldehyde was obtained from Electron Microscopy Sciences (Ft. Washington, PA). Laboratory rodent diet (Purina Laboratory Diet, 5001) containing 13 ppm copper and the same diet supplemented to 200 ppm copper using copper sulfate were obtained from Purina Mills (Richmond, IN). CS2 was obtained from EM Sciences (Gibbstown, NJ), and pyrrolidine was purchased from Alfa Aesar (Ward Hill, MA).
Chemical Syntheses
Pyrrolidine dithiocarbamate.
Freshly distilled pyrrolidine (14.25 g, 0.2 mole) was taken in a mixure of ethanol (125 ml) and water (75 ml), and stirred in an ice bath. Carbon disulfide (15 ml, 0.25 mole) was added, followed by 10 N NaOH (20 ml). After 1 h the precipitated solid was washed with absolute ethanol and dried; 30 g (88%). The identity and purity (>99%) of pyrrolidine dithiocarbamate was verified by NMR spectroscopy and by UV Spectrophotometry.
S-(Piperidin-1-ylcarbonyl)cysteine.
S-(Piperidin-1-ylcarbonyl)cysteine (Pip-cys) was synthesized as previously described (Zimmerman et al., 2004). TEAB-ACN buffer was prepared from 1 M triethylammonium bicarbonate (pH 8.0, 2 ml), triethylamine (0.5 ml), water (50 ml), and acetonitrile (50 ml).
Animals and exposures.
All exposures were conducted in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee. Male Sprague-Dawley rats obtained from Harlan Bioproducts (Indianapolis, IN) were given food and water ad libitum and caged in a room on a diurnal light cycle. Body weights were determined prior to the start of the study and then weekly during the course of the experiment. Four exposure groups were used: two PDTC-exposed groups receiving 8 mM PDTC in deionized water and two control groups given tap water. One control group (n = 8) and one PDTC group (n = 8) were given Purina rodent diet 5001 containing 13 ppm copper (standard rat maintenance diet fed at Vanderbilt University), and the remaining control group (n = 8) and PDTC group (n = 8) were given Purina rodent lab diet 5001 containing 200 ppm copper. The 200 ppm copper diet was chosen for copper loading after literature review indicated no significant clinical, chemical, histopathological, and hematological toxicity at this level when chronically administered (Cristofori et al., 1992; Everling et al., 1990; Fuentealba et al., 2000; Milanino et al., 2000), while still affording a 15-fold increase in dietary copper. The average starting weight of the 13 ppm copper diet animals was 291 ± 3 g, and the average starting weight of the 200 ppm copper diet animals was 401 ± 6 g. PDTC was delivered as an aqueous solution of NaPDTC in deionized water, the concentration of which was determined from the UV absorbance at 277 nm ( = 13,540 M–1 cm–1). The dosing of PDTC in the drinking water of both normal copper and high copper treatment groups was accomplished gradually (to avoid rejection of the solution) by increasing the concentration from 4 mM to 8 mM over a period of 21 days. This was the maximum oral tolerated dose. Water intake between groups was compared at the beginning of week 16 of the study and was quantified by measurement of water intake per cage (four animals per cage) over a 10-day period. Four of the 13 ppm copper PDTC and 13 ppm copper control rats were analyzed first at 18 weeks to look for differences in tissue copper levels and to detect early peripheral nerve lesions, and the remaining 13 ppm copper animals were taken out to 58 weeks. Three of the 200 ppm copper PDTC and 200 ppm copper control rats were analyzed at 47 weeks when three animals in the 200 ppm copper PDTC group had to be removed from the study due to significant body weight loss and paresis of their hindlimbs, and at that time, one other 200 ppm copper PDTC animal with the lowest grip strength was sacrificed along with matched controls. The remaining 200 ppm copper PDTC and control animals were continued to 58 weeks when significant paresis and weight loss was evident. At each time point examined, neuromuscular function was assessed by measuring hindlimb grip strength using a digital force gauge (Meyer et al., 1979), and tissues were collected for analyses (see below).
Blood collection.
After induction of deep anesthesia, whole blood (3–4 ml) was collected from the left ventricle prior to perfusion or tissue collection. A volume of 0.6–1.0 ml whole blood was placed into a heparinized Eppendorf tube for globin isolation and subsequent analysis by HPLC or LC/MS/MS; and 2–3 ml was allowed to clot in serum separator tubes, and serum was collected after centrifugation for copper analysis by RP-HPLC (see below). Heparanized blood was separated into plasma and hemolysate as previously described (Erve et al., 2000), and 100 μl of 1 M ascorbic acid was added to 1 ml of hemolysate. The resulting solution was added dropwise to 7 ml of 2.5% oxalic acid in acetone. Globin was allowed to precipitate on ice for 15 min, and then centrifuged at 10,000 x g for 10 min at 4°C, washed in 5 ml ice cold acetone, and centrifuged at 10,000 x g for 10 min. The resulting pellet, containing crude globin, was dried under a nitrogen stream and stored at –80°C. Dried globin was solubilized with 0.1% trifluroacetic acid (TFA) to produce a solution for HPLC analysis.
Globin chains were separated by RP-HPLC on a Phenomenex Jupiter 5-μm, 300 column (150 x 460 mm) using a Waters 2690 liquid chromatograph after adjusting sample concentration to a UV absorption of 1 at 280 nm. Globins were separated using a linear gradient from 56:44% (Solvent A:Solvent B) to 30:70% (A:B) over 30 min, followed by a linear gradient to 100% solvent B (1 min.) holding for 5 min, reequilibrating to 56:44 (A:B) over 10 min, and holding for 10 min. Solvent A was 20:80:0.1 acetonitrile:water:TFA, and solvent B was 60:40:0.08 acetonitrile:water:TFA. The elution of globin peaks was monitored by their UV absorption at 220 nm using a Waters 996 photodiode array detector.
Preparation of nerve tissue and morphological assessments.
Animals examined at 18 weeks were perfused through the left ventricle of the heart under deep anesthesia (100 mg/kg ketamine HCL plus 15 mg/kg xylazine) with a solution of 0.8% NaCl, 0.025% KCl, 0.05% NaHCO3, in 0.01 M phosphate buffer followed by 4% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. Animals examined at 47 and 58 week timepoints were exsanguinated under deep anesthesia and not perfused. After exsanguination or perfusion, liver, brain, and sciatic nerves were dissected out and immersed in 4% glutaraldeyde in 0.1 M phosphate buffer overnight and then transferred to 0.1 M phosphate buffer (pH 7.4). Sciatic nerve sections were post-fixed with osmium and embedded in Epon; thick sections were cut and stained with toluidine blue. Thick (1 μm) sections of peripheral nerve tissue were evaluated by light microscopy on an Olympus BX41 microscope equipped with an Optronics Microfire digital camera. Four different fields at 40x (total area = 0.26 mm2) were photographed from one section of each sciatic nerve, and the total number of lesions counted by one observer (HLV). The lesions quantified were: degenerated axons, axons with thin myelin (myelin sheath with an axon/fiber diameter ratio (g ratio) of >0.7 (King, 1999)), intramyelinic edema, and demyelinated axons. Thin (70 nm) sections were prepared from sciatic nerves and evaluated using a Phillips CM-12 electron microscope, 120 keV with a high resolution CCD camera system.
Analysis of tissue metal levels.
Tissue sections of brain, liver, and sciatic nerve were analyzed by inductively coupled plasma-atomic emission spectroscopy (ICP-AES) at the Diagnostic Center for Population and Animal Health at Michigan State University (East Lansing, MI). Elements determined in this analysis were copper, calcium, iron, and zinc. Individual liver and brain samples were analyzed in duplicate for mineral content, and the results reported as one value per individual. One sciatic nerve was taken from each animal and pooled with other members of the same treatment group to provide adequate tissue for ICP-AES analysis, and this resulted in one analysis injected twice per group, thus generating two values per group. Brain tissue samples for ICP-AES were approximately 2-mm midsagittal slices; liver sections were approximately 1-cm3 pieces of tissue; sciatic nerve sections were approximately 15- to 20-mm lengths of a single nerve.
Analysis of serum copper levels.
Serum (100 μl) was mixed with 6 M HCl (20 μl) at ambient temperature for 10 min before cooling in ice and centrifugation (13, 000 x g) for 16 min. An aliquot (80 μl) of the supernatant was neutralized with 20 μl 1 M K2HPO4 and 30 μl 4 M NH4OH, and treated with 40 μl of 20 mM bis-hydroxyethyl dithiocarbamate (HEDC). After 10 min the copper complex was extracted with ethyl acetate (2 x 400 μl), solvent evaporated under a stream of nitrogen, and residue dissolved in 50 μl of 60:40 10 mM ammonium acetate-acetonitrile. The solvent mixture of 60:40 water:acetonitrile was used for analyzing 5 μl of the reconstituted solution by HPLC on an Alltex Alltima HP C18 reversed-phase column (2.1 x 150 mm; 5 μm). A photo-diode array detector in the wavelength range 360 to 460 nm was used to monitor the elution, and absorbance at 430 nm was used for measurements. A standard curve using known concentrations of Cu-HEDC from 0 to 250 μM was generated.
LC/MS/MS analysis of globins.
Globin samples (5–10 mg) were hydrolyzed with 6 M HCl for 4 h, and excess acid was removed. Internal standard (5 μl of 100 μM Pip-cys) was added to the residue, which was dissolved in TEAB-ACN buffer (200 μl), and triethylamine (10 μl), and 3-pyridylisothiocyanate (40 μl of a 50 μM solution in ACN) were added to this. The mixture was agitated in a water bath at 37°C for 1 h and evaporated. The residue was reconstituted with 10 mM HCl (100 μl), and a 10 μl aliquot was analyzed by LC/MS/MS. An Exterra MS C18 column (2.1 x 100 mm; 3.5 μm) running at 0.2 ml/min was used. A gradient elution from 100% A (10% methanol in 5 mM formic acid) to 60% B (100% methanol in 5 mM formic acid) over 20 min was used to elute the analyte and the standard. Selected reaction monitoring (SRM) using the daughter ion m/z 137 generated from the molecular ions 355 (PDC-Cys adduct) and 369 (Pip-Cys adduct) was used for measurements. Known amounts of PDC-Cys and Pip-Cys were taken, dried, derivatized, and analyzed by SRM. Mole ratios of the two were plotted against area ratios to generate a standard curve. Amounts of PDC-Cys were estimated using the calculated response factor.
Statistical analysis.
One-way analysis of variance (ANOVA) with Tukey-Kramer's Multiple Comparisons Test and the one-tailed t-test were performed using GraphPad Software, Inc. Lesion counts were square root transformed to normalize their distribution and equalize variances prior to statistical comparisons by ANOVA and Tukey-Kramer's Multiple Comparisons Test. For two group comparisons where equal variances were not obtained as determined by the F test, the t-test corrected for unequal variance was used. Statistical significance was taken to be p < 0.05 unless otherwise noted.
RESULTS
Body Weights and Dosage Levels
Administration of PDTC resulted in significantly reduced body weights relative to controls at each time point (Table 1). Since at the 58-week time point, the body weights of same strain male animals were expected to be similar in the absence of dietary copper manipulations or PDTC administration, all groups were compared regardless of average starting weights. No significant effect on body weight from increased dietary copper alone was apparent.
The average dose of PDTC taken in the water was determined by measuring water volume intake every 2 days per group over 10 days beginning at week 16. A value of 10.5 ml of 8 mM PDTC/100 g BW value was obtained on rats given PDTC eating a 13 ppm copper diet, and an average of 11.0 ml of 8 mM PDTC/100 g BW was obtained on rats given PDTC eating a 200 ppm copper diet. This was calculated to be 0.85 mmol/kg/day for PDTC-treated, 13 ppm copper diet animals and 0.88 mmol/kg/day for PDTC-treated, 200 ppm copper diet animals.
Analysis of Tissue Copper Levels
Tissue copper levels for each time point are presented in Figure 1. Brain copper levels in all PDTC-exposed groups were significantly elevated over same-diet controls. As the study progressed from 18 to 58 weeks, brain copper levels increased in both the control and PDTC-treated rats, with those in the combined PDTC and high-copper diet containing the greatest levels. Similar to brain, sciatic nerve copper was also the greatest in the groups on the combined PDTC high-copper diet, and sciatic nerve copper appeared to accumulate as a function of age in controls, with more accumulation occurring in the high copper diet controls.
In contrast, liver copper was only significantly increased in PDTC-exposed animals that also received the 200 ppm copper diet at the 47 and 58 week time points.
The only significant difference noted for serum copper levels that appeared to be dithiocarbamate-related was an increase in the 47-week PDTC 200 ppm copper group relative to the 47-week control 200 ppm copper group.
Analysis of Tissue Calcium, Iron, and Zinc
Although there were some significant differences between treatment groups at all three timepoints, there were no trends with any of the three minerals consistent with compound-related changes. These results are presented as supplementary data.
Globin Analyses
Additional late-eluting peaks resulting from covalent modification of the globin 3 chain analogous to those produced by parenteral DEDC administration were not detected in any of the PDTC-dosed rats at either 18, 47, or 58 weeks.
The greater sensitivity afforded by LC/MS/MS analysis made it possible to demonstrate the presence of PDC-Cys adducts on globin (Erve et al., 2000; Tonkin et al., 2000, 2003). At 58 weeks, the level of PDC-Cys on globin for 200 ppm PDTC-exposed animals was significantly greater than for PDTC, 13 ppm copper diet animals and both control groups (Table 2).
Neuromuscular Function and Peripheral Nerve Morphology
Significant decreases in grip strength were observed at each timepoint for PDTC-dosed rats relative to their respective controls (Table 3). In addition, administration of the high-copper diet with PDTC produced a significant decrease in grip strength relative to PDTC with the 13 ppm copper diet. No difference in grip strength resulted from 200 ppm copper diet alone relative to controls on the 13 ppm copper diet.
Representative sections of sciatic nerve obtained from controls and animals exposed to PDTC demonstrating the lesions quantified in Table 4 are shown at the light microscope level in Figure 2 and electron microscope level in Figure 3. Peripheral nerve lesions were observed both in animals exposed to PDTC alone and in animals exposed to PDTC in combination with a high-copper diet that were not present in control animals on either diet. A significant increase in axons exhibiting thin myelin and undergoing degeneration was observed in the 18-week, PDTC group. At 47 weeks, two animals in the PDTC 200 ppm copper group had become very weak in their hind legs and had lost a substantial amount of body weight. They were removed from the study along with one other PDTC, 200 ppm copper rat with the next lowest grip strength along with three 200 ppm copper controls, and their tissues were collected. The two most affected PDTC, 200 ppm animals exhibited much higher numbers of degenerated axons, thinly myelinated axons, intramyelinic edema, and demyelinated axons than the other affected PDTC, 200 ppm animal, and the number of demyelinated and degenerated axons in the PDTC 200 ppm copper group was significantly elevated over the 200 ppm controls. At 58 weeks, the remaining PDTC 200 ppm copper-exposed animals were clinically weak and had significant weight loss. Also at 58 weeks, a significant increase in thinly myelinated axons, intramyelinic edema, and degenerated axons was seen in the PDTC 200 ppm copper animals as compared to the PDTC 13 ppm copper animals, the 200 ppm controls, and the 13 ppm controls. Also a significant increase in demyelinated axons was seen in the PDTC 200 ppm copper animals as compared to the 200 ppm and 13 ppm controls. Axonal regeneration and remyelination was evidenced by the presence of axonal sprouts and onion bulbs, respectively.
DISCUSSION
Numerous biological effects have been reported for pyrrolidine dithiocarbamate in vitro and in vivo including pro-oxidant, antioxidant, chelation, antiprotozoal, antiviral, and antineoplastic effects, but relatively little information is available regarding its potential neurotoxicity in vivo.
Due to the pyrrolidine ring structure, PDTC is substantially more resistant to acid-promoted decomposition to parent amine and CS2 than noncyclic aliphatic dialkyl dithiocarbamates, e.g., DEDC, and this has been the principal reason for using it instead of DEDC within biological systems. The relative acid stability of PDTC suggests that a greater percentage of an oral dose would be absorbed intact as parent dithiocarbamate relative to DEDC. Consistent with this interpretation, there was no evidence for CS2-mediated intramolecular protein cross-linking of the -globin chain observed by HPLC in this study. Thus, if parent dithiocarbamate is the proximate toxic species for the myelin lesions as previously proposed, PDTC would be predicted to be a Schwann cell toxicant similar to disulfiram and parenteral DEDC. This was indeed the case, and the structural changes produced in peripheral nerve by oral PDTC corresponded to a chronic demyelinating injury, evidenced by naked axons, onion bulb formation, intramyelinic edema, and thinly myelinated axons. Axonal degeneration was also observed. In many cases this appeared to be a secondary axonal degeneration subsequent to Schwann cell injury, but the observation of axonal degeneration at the earliest time point suggests that there may be some primary axonal involvement, although thinned myelin was also a significant finding at this time point.
Although carbamylation of proteins by dithiocarbamates does not appear to underlie the demyelinating injury produced by dithiocarbamates (Tonkin et al., 2003), it has been used as an internal dosimeter and for monitoring the distribution of dithiocarbamates in vivo (Erve et al., 2000; Tonkin et al., 2004; Zimmerman et al., 2004). Additionally, cysteine carbamylation is thought to be responsible for inhibition of aldehyde dehydrogenase, which is the basis for using disulfiram in alcohol aversion therapy (Shen et al., 2001; Zimmerman et al., 2004). In the current study, analysis of globin samples by LC/MS/MS revealed that PDTC also carbamylates cysteine residues on globin, but to a much lower extent than DEDC or disulfiram, as evidenced by HPLC. For PDTC there were no readily identifiable new HPLC peaks in globin preparations, whereas neurotoxic doses of parenteral DEDC and oral disulfiram (Tonkin et al., 2000, 2003) have been observed to modify up to 13 to 24% of the chains of globin. At least two interpretations for this finding are possible. Modification of Cys-125 on the 3 chain of globin by PDTC may not alter its retention time to the same extent as DEDC, or more likely, PDTC is not metabolized to a reactive sulfoxide or sulfone intermediate to the same extent as DEDC.
Although zinc, iron, and calcium were also measured in tissues, no trends were noted like those seen with copper. This is consistent with a previous report (Calviello et al., 2005), where an elevation in tibial nerve copper occurred without changes in zinc or iron following oral administration of PDTC. This may be due to the formation of unstable PDTC chelates with these other ions, rendering them unable to enter lipid-containing tissues. Interestingly, the 58-week PDTC 200 ppm Cu group had significantly greater severity scores for most categories compared to the other 58-week groups, including the PDTC 13 ppm copper group. These data, together with the elevated copper levels present in the sciatic nerves of the PDTC high-copper diet animals, suggest that disruption of copper homeostasis by PDTC may contribute to the development of lesions in peripheral nerve and that PDTC is able to do this through the formation of a nonionic chelate with Cu2+ which can cross the blood–brain barrier and accumulate in lipid-containing tissues. Additionally, oral administration of PDTC increased the level of copper in brain and had negative effects on weight gain and hind limb grip strengths. Similar to the situation observed for peripheral nerve lesions, dietary copper supplementation also enhanced the effects of PDTC for these three parameters. In contrast, elevations in liver and serum copper were only observed in the POTC groups fed the high-copper diet. Copper levels in sciatic nerve were also clearly elevated in the groups administered both PDTC and the high-copper diet, but the effect of PDTC alone was less clear. Although there appeared to be a trend for PDTC to increase copper in sciatic nerve at the early time point, this trend was not verified at 58 weeks. Since elevations of brain copper produced by PDTC with the normal copper diet paralleled previous results reported for DEDC, and DEDC is able to increase sciatic nerve copper without increased dietary copper, the values obtained for sciatic nerve copper levels in the PDTC normal copper diet animals at 58 weeks may be anomalous. Unfortunately the quantity of sample needed for ICP-AES required pooling of the nerves, decreasing the n and preventing reanalysis of the samples. Therefore, further investigation will be required to unequivocally resolve this question.
The findings of the present study are consonant with previous studies in which routes of exposure and chemical properties that favored absorption of intact dithiocarbamate with copper accumulation in the nervous system were associated with myelinopathy. Additional evidence was obtained that the level of copper accumulated by a dithiocarbamate may be positively correlated to the severity of injury within peripheral nerve. Considering the tight regulation of copper within biological systems and the extremely limited amount of free copper (Rae et al., 1999), the magnitude of changes in copper levels observed here could have substantial biological effects if only a very small fraction of the increased copper existed as free copper. Indeed, previous studies have suggested a role for copper-promoted oxidative stress in dithiocarbamate-mediated neurotoxicity based upon associated elevations in markers for lipid peroxidation (Delmaestro and Trombetta, 1995; Tonkin et al., 2004). It should be noted, though, that these analyses were performed at advanced endpoints and, at least in the case of peripheral nerve, were accompanied by substantial injury. This raises the possibility that the elevation of lipid peroxidation products observed might not be a specific finding for the dithiocarbamate, but possibly a more general consequence of the ongoing demyelinating process accompanied by the influx of active macrophages. In contrast, evidence has also been provided that deficiencies of copper can result in myelinopathies (Dake and Amemiya, 1991; Zimmerman et al., 1976), and the potential for dithiocarbamates to sequester copper and limit its availability as a cofactor for certain enzymes (Heikkila et al., 1976), e.g., copper zinc superoxide dismutase and dopamine hydroxylase, presents an alternative hypothesis. But the more severe lesions obtained for the high-copper diet animals appears contradictory to this interpretation. To ascertain the role of dithiocarbamate-mediated alteration of copper homeostasis in peripheral nerve injury, further investigation is required. Studies directed at determining the chemical species responsible for copper elevation in nerve, defining the distribution of elevated copper in nerve and delineating the temporal relationship of oxidative injury to the onset of structural changes will provide useful information toward this goal. The significance of defining the role of copper accumulation in dithiocarbamate neurotoxicity could be realized through facilitating the development of structure–activity relationships useful for defining the relative risks associated with various dithiocarbamates and designing safer dithiocarbamates.
SUPPLEMENTARY DATA
Zinc, iron and calcium levels measured by ICP-AES for liver, brain and sciatic nerve tissue are provided as supplementary data in a word document named Zn, Fe, Ca suppdata.doc. Supplementary data are available online at www.toxsci.oxfordjournals.org.
ACKNOWLEDGMENTS
The authors would like to thank Dr. Doyle Graham for his consultation and mentoring of peripheral nerve pathology. NMR analysis of pyrrolidine dithiocarbamate was performed by Dr. Lisa Zimmerman. LC/MS/MS analysis was performed with the assistance of Lisa Manier of the Mass Spectrometry Research Center at Vanderbilt University. Experiments were performed in part through the use of the VUMC Research EM Resource (sponsored by NIH Grants DK20539 and DK58404). This work was supported by NIEHS Grant ES06387 and by the Center of Molecular Toxicology Grant P30 ES00267.
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