Protective effects of glutathione on diethyldithiocarbamate (DDC) cytotoxicity: A possible mechanism

93, 154-164(1988)

TOXICOLOGYANDAPPLlEDPHARMACOLCCY

Protective

Effects of Glutathione on Diethyldithiocarbamate Cytotoxicity: A Possible Mechanism

LOUIS D. TROMBETTA,’ MAUREEN TOULON, Toxicology

Program,

ReceivedApril

St. John’s

University,

16,1987;

Jamaica,

acceptedNovember

(DDC)

AND I. SIRAJ JAMALL New York

11439

5,1987

Protective Effects of Glutathione on Diethyldithiocarbamate (DDC) Cytotoxicity: A Possible Mechanism. TROMBETTA, L. D., TOULON M., AND JAMALL, I. S., (1988). Toxicol. Appl. Pharmacol. 93, 154- 164. Rat cerebral astrocytes grown in culture were exposed to 35 rg diethyldithiocarbamate (DDC)/ml of medium for I hr and treated with 0 or 10 mM reduced ghrtathione (GSH) 1 hr post-DDC. DDC treatment resulted in a 90% reduction in cell adherence within 24 hr and complete inhibition of growth. The most pronounced ultrastructurai lesion in DDCtreated cells was on mitochondria. Numerous lipofuscin-like deposits were seen in these cells. In addition, DDC treatment resulted in a greater than 400% increase in cellular copper. The activity ofthe selenoenzyme ghttathione peroxidase was reduced by about 40% with no concomitant effect on cytosolic superoxide dismutase activity. The data suggest that DDC cytoxicity is peroxidative in nature, presumably due to the massive influx of copper into the astrocyte. GSH treatment 1 hr after exposure ofthe cells to DDC completely prevented the DDC-induced reduction in cell adherence and growth inhibition. Ultrastructurally, cells post-treated with GSH prevented much ofthe damage caused by DDC. This protection was associated with marked reduction in cellular copper and a return to control glutathione peroxidase activity. o 1988 Academic Press, Inc.

Diethyldithiocarbamate (DDC), a potent metal chelator, is used in the treatment of Wilson’s disease (Sunderman et al., 1963) and of nickel poisoning (Sunderman, 197 1). Currently, clinical use of dithiocarbamates is limited to disulfiram (Antabuse) which is used to induce alcohol aversion through inhibition of aldehyde dehydrogenase (Sellers et al., 198 1; Sanny and Weiner, 1987). Disulfiram is rapidly reduced to DDC in vivo by reaction with glutathione (GSH), a major physiological reducing agent found in cytosol, mitochondria, plasma, and bile (Stromme, 1965a, 1966). Evidence also exists for the limited reoxidation of DDC to disulfiram at the cellular level (Inoue et al., 1982; Forman et al., 1980; Stromme, 1965b).

’ To whom all correspondence should be addressed, 0041-008X/88

$3.00

Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

Both disulfiram and DDC have been reported to be neurotoxic (Trombetta and Adachi, 1985; Mokri et al., 1981; Hotson and Langston, 1976). The pathogenesis of this toxicity is not known. Puglia and Powell ( 1984) have suggested that impairment of cellular antioxidant activity may be the primary cause of toxic cell injury. The ability of DDC to impair the activity of one of the components of the antioxidant machinery, coppermodulated cytosolic superoxide dismutase (SOD), is thought to be a significant factor in DDC-induced CNS toxicity (Heikkila et al., 1976; Puglia and Loeb, 1984). The reactivity of disulfiram and DDC with suhhydryl groups may also impair the antioxidant activity ofglutathione (Goldstein et al., 1979; Sunderman et al., 1984). Aaseth et al. (198 1) have suggested that DDC-induced CNS toxicity may be related to 154

GSH AND DIETHYLDITHIOCARBAMATE

the ability of DDC to preferentially enhance the redistribution of endogenous copper to the brain. CNS lesions associated with increased copper levels have been reported in the brains of rabbits (Edington and Howell, 1966) and lambs (Howell et al., 1970) after long-term treatment with DDC. Work in our laboratory (Trombetta and Adachi, 1985) has demonstrated that rats chronically treated with disulfiram exhibit degenerative changes in astrocytes prior to alterations seen in neurons. Thus, the present study employed a astrocyte cell culture system to elucidate the mechanism of DDC cytotoxicity with particular emphasis on the role of the cellular antioxidant defense system in preventing or ameliorating DDC-induced injury. METHODS Cell Culture Systems Cerebral cortical astrocytes from male Sprague-Dawley rats were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing glutamine and 4.5 mg glucose/liter, supplemented with 10% heat-inactivated fetal bovine serum (FBS), streptomycin, 0.1 mg/ml, and penicillin 100 U/ml on Primaria flasks in a mixture of 8% CO2 and 92% air. Cu levels in the medium were found to be 0.198 f 0.0 16 ppm by analysis in pentuplicate. Viability was determined by trypan blue exclusion. Astroglial lineage of these cells was confirmed by their reactivity to glial fibrillary acidic protein (GFAP) using a peroxidaseantiperoxidase labeling technique (Ludwin et a/., 1976). Cells grown in go-cm* flasks were exposed to 35 jrg DDC/ml (1.5 X 10m4M DDC) for 1 hr. After rinsing with medium, flasks were drained and post-treated with complete medium containing either 0 or 10 mM GSH for 24 hr. The medium for these GSH post-treated cells was then drained, rinsed and replaced with fresh non-GSHsupplemented medium. Other protocols included (a) pretreatment with 10 mM GSH in complete medium for 1 and 15 hr prior to DDC (35 pg DDC/ml) exposure, and (b) simultaneous exposure of the cells for 1 hr to a premixed solution (for 10 min) of 35 pg DDC/ml medium and 10 mM GSH for 1 hr. In all experiments, the responses of the DDC-treated cell populations were compared to control cells of the same passage. Cell Adherence After treatment cells were passed into duplicate 25cm’ flasks at a density of 5 X 10’ cells/flask. After 24 hr

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the medium from each flask was collected and the flasks were rinsed twice with 5 ml phosphate-buffered saline (PBS). For each flask, the medium and PBS rinses were combined and centrifuged. The cells collected in this manner were counted as nonadherent cells. The adherent cell population was collected by trypsinization and counted. The percentage adherence was determined.

Growth Curve Cells treated as described above were resuspended in complete medium at a density of 1.25 X 1O5cells/flask (3 ml medium/flask on Day 0). Eighteen hours after explantation, the medium in all flasks was replaced with 4 ml fresh complete medium. The average cell count of three flasks from each group was recorded on Days 1 through 6. Cell viability was measured by trypan blue exclusion.

Electron Microscopy Cells from the various groups were fixed with phosphate-buffered (pH 7.4) glutaraldehyde (1.5%) for 1 hr at room temperature and overnight at 4’C. The cells were washed in buffer and postfixed in phosphate-buffered (pH 7.4) osmium tetroxide (1 .O%) at 4’C. Tissue was processedby standard procedures, embedded in an Epon Araldite mixture, and viewed at 75 kV on a Hitachi HU 11E electron microscope.

Biochemicaf Assays Since control cells for each cell passage varied in the amount of analyte, the percentage differences between controls and treated cells were studied statistically. The average quantity of a particular analyte in control cells is given only to serve as a frame of reference for this cell line. Glutathione peroxidase (GSH-Px). The activity of the selenoenzyme GSH-Px was measured according to a modification of the method of Germain and Arneson (1977). The modifications included the incorporation of 1 mM NaN3 into the reaction mixture to inhibit catalase activity and the addition of 0.2 mM NADPH instead of 0.1 mM NADPH. The reaction was initiated by the adding 0.1 ml H202 instead of cumene hydroperoxide to give a final concentration of 0.16 mM HZ02. This was done to ensure the measurement of the selenoenzyme only. Superoxide dismutase. After trypsinization, the cells were washed twice with PBS (pH 7.4) containing 3 mM diethylenetriaminepentaacetic acid (DTPA). DTPA is a chelator that prevents reactivation of cytosolic SOD by copper ions (Westman and Marklund, 1980). SOD was measured by the method of Winterboum ef a/. (1975). Additional experiments were performed to evaluate the

156

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AND JAMALL

RESULTS Efects of DDC Growth

FIG. 1. Cell adherence: Effect of DDC and post-treatment with 10 mM GSH on cell adherence after 24 hr. After 1 hr exposure to DDC cells were rinsed with medium and then incubated with 10 mM GSH through 24 hr.

effectsof higher doses of DDC (350 and 700 pg DDC/ml of medium) on SOD levels. GSH and GSSG levels. Fluorometric measurements (Gilford Fluoro IV spectrofluorimeter) of GSH and GSSG were determined by a modification of the method of Mokrasch and Teschke (1984) with the addition of Nethylmaleimide (NEM) for measurement ofGSSG as described by Hissin and Hilf (1976). Protein determination. The protein concentration of all samples was measured by the Coomassie brilliant blue G-250 assayof Bradford (I 976) using bovine serum albumin as a standard. Metal Analysis

on Cell Adherence

and

As shown in Fig. 1 exposure of the astrocytes to 35 pg DDC/ml medium for 1 hr caused a 90% reduction in cell adherence within 24 hr. Virtually all cells in this treatment group were nonadherent after 48 hr and remained so throughout the experiment. Addition of 10 IIIM GSH after DDC treatment completely prevented this effect of DDC. Cells pretreated with GSH prior to DDC administration and cells treated simultaneously with GSH and DDC exhibited results similar to those of cells treated with DDC alone. Thus, only post-treatment with GSH protected against DDC toxicity. Figure 2 shows the effect of DDC exposure, with and without post-treatment with GSH, on the normal growth cycle of rat astrocytes. Complete inhibition of the proliferative response was seen in the cell populations treated with DDC alone. However, cells in this group remained viable until Day 6. Cells treated with 10 mM GSH 1 hr after exposure to DDC exhibited a growth pattern similar to that of control cells. Viability of the control group and the GSH post-DDC group remained at more than 99% throughout the experiment.

Drained cell pellets were digested in 10% perchloric acid (PCA) at a density of IO6cells/ml. Further dilutions, where necessary, were carried out with deionized water. Medium from treatment flasks was prepared for metal analysis by the addition of PCA to give a final concentration of 10% PCA. Copper was determined using a Perkin-Elmer Model 2380 atomic absorption spectrophotometer against known standards (Jamall and Sprawls. 1987). Siatistical Analysis Significance was tested between one of the treated groups and untreated controls at each time interval using the one-way analysis of variance (ANOVA) followed by the Tukey post hoc analysis to test for difference among several means. Significance values less than 0.05 were considered to be significant. Data are presented as a percentage of control values.

o**s FIG. 2. Growth curve: Effect of DDC and post-treatment with GSH on cell growth. Each point represents the mean cell count ofthree replicate flasks.

GSH AND DIETHYLDITHIOCARBAMATE

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FIG. 3. Electron micrograph of a control rat astrocyte. Notice the large number of polyribosomes (small arrows). The rough endoplasmic reticulum appears as short strands containing a floccular material within its cistemae (curved arrow). Nucleus (Nu).

Electron Microscopy Control astrocytes appeared either stellate or polygonal in form with some cells containing long processes. Electron microscopy showed cells which were uniform in their cytoplasmic and nuclear detail (Fig. 3). These cells contained rounded nuclei with irregular, convoluted surfaces and normal nucleoli. Numerous small, smooth-surfaced vesicles were seen scattered throughout the cytoplasm. Rough endoplasmic reticulum (RER) appeared as short strands, sometimes dilated, containing a floccular material within their cisternae. Numerous polyribosomes were seen throughout the cytoplasm. The mitochondria appeared normal with clearly discerned cristae. Occasionally a myelin whorl was seen in the cytoplasm. The most marked effect seen in cells treated with DDC was the pronounced alter-

ations in mitochondrial structure (Fig. 4). Distortion was seen in the organization of their membranes, leading to swelling, rupture, and increased lucency of the matrix. Cytoplasmic clearing and rupture of the plasmalemma was also observed as were decreases of RER. Markedly dilated vesicles either derived from the RER or Golgi apparatus were also seen. They often appeared lucent but sometimes contained particulate material. Inclusion bodies similar to myelin whorl formation and lipofuscin were present in the cytoplasm of the most severely affected cells. Non-membrane-bound lipid droplets were also evident in these severely affected cells. Not all cells treated with DDC showed the severe changes described above. This may be in accord with the growth curve data in which many cells, although unable to proliferate or adhere, remained viable for several days after

158

TROMBETTA,

TOULON.

AND JAMALL

FIG. 4. Electron micrograph of a rat astrocyte after exposure to 35 Kg DDC/ml medium. Note the distorted mitochondria (small arrows), vacuoles (v). and altered vesicles of the Golgi apparatus (curved arrows). Note the large lysosome-like structures in the cytoplasm (large arrows). These structures probably represent residual bodies. However, some appear to have a double membrane and may represent disrupted mitochondria.

DDC exposure. The most severe changes exhibited by these cells were in the mitochondria and were similar to alterations described above. The Golgi apparatus and ER were absent but numerous polysomes were seen in the cytoplasm. Dilated vacuoles were also seen in the cytoplasm. These may represent rough endoplasmic reticulum since ribosomes were sometimes seen on their membranes. Fewer inclusion bodies were seen in these less affected cells (Fig. 5). Cells treated with DDC and post-treated with GSH showed remarkable similarity to normal cells with the exceptions noted below. The most obvious difference between these cells and controls was the presence in the cytoplasm of numerous residual bodies and lipid droplets. This was similar to what was seen in DDC-treated cells. They also contained numerous dilated vesicles and some

electron-lucent mitochondria. The Golgi apparatus appeared very active and, at times, gave the appearance of a dictyosome (Fig. 6). Biochemical

Efects

Cu levels. Table 1 summarizes the effect of DDC on cellular Cu. Exposure to DDC resulted in a greater than 400% increase in cellular Cu content. With post-treatment with GSH for 24 hr Cu levels returned to near control values. GSH-Px activity. DDC reduced GSH-Px activity by approximately 40% within 4 hr of treatment. By 24 hr, activity had returned to 76% of control levels. Post-treatment with 10 mM GSH brought GSH-Px activity to 79% of control levels at 4 hr and to control lelrels by 24 hr (Table 2).

GSH AND DIETHYLDITHIOCARBAMATE

CYTOTOXICITY

159

FIG. 5. Electron micrograph of a rat astrocyte after exposure to 35 pg DDC/ml medium. Note that this cell does not contain the lysosomal structures seen in the previous micrograph. Mitochondria (M) in these cells also appeared abnormal. Cytoplasmic disruption and membrane alterations (open arrow) could be seen. Polyribosomes can be seen in the cytoplasm (small arrows) and dilated vacuoles (curved arrow). Nucleus (Nu).

SOD activity. Treatment of astrocytes with 35 pg DDC/ml medium did not significantly affect cytosolic SOD after 4 and 24 hr (Table 3). A tenfold higher DDC concentration (350 pg DDC/ml) was required to elicit a mere 7% reduction in SOD activity and only a concentration of 700 pg DDC/ml effected a 52% reduction in cytosolic SOD activity (data not shown). GSH and GSSG levels. Relative to control populations of the same cell passage, no significant change in GSH could be demonstrated 4 and 24 hr after treatment with DDC. Cells exposed to DDC and then post-treated with GSH exhibited 49% higher GSH levels at 4 hr. This increase in cellular GSH returned to normal at 24 hr. Cells treated with GSH alone exhibited a 17 1% elevation and a 130% elevation in GSH levels at 4 and 24 hr, respectively (Table 4).

No significant alteration in cellular GSSG levels were observed in DDC-treated astrocytes at 4 or 24 hr. DDC-treated cells that received 10 mM GSH post-DDC exhibited a 243% increase in GSSG at 4 hr and a 194% increase in GSSG at 24 hr relative to untreated controls. These GSSG levels were not significantly different from those observed in cells treated with GSH alone (Table 4). DISCUSSION These data indicate that the mechanism of DDC toxicity to astrocytes in culture involves a marked increase in the Cu content of these cells. Furthermore, exposure to DDC (35 pg/ ml for 1 hr) results in a 40% decrease in the activity of the selenoenzyme GSH-Px. SOD activity was unaffected at this DDC concen-

160

TROMBETTA,

TOULON.

AND

JAMALL

FIG. 6. Electron micrograph of a cell exposed to 35 fig DDC/ml medium and post-treated with 10 rnM GSH for 24 hr. Note the large amounts of rough endoplasmic reticulum (er) and free ribosomes. An active Golgi apparatus is also seen. These cells contained numerous vacuoles(v), lipid droplets(L), myelin figures (open arrows), and inclusion bodies (large arrows). Nucleus (Nu).

GSH

AND

TABLE

DIETHYLDITHIOCARBAMATE

161

CYTOTOXICITY

1

TABLE

3

EFFECTSOF DIETHYLDITHIOCARBAMATE (DDC) AND GLUTATHIONE (GSH) ON COPPER LEVELS IN ASTROCYTESIN VITRO’

EFFECTSOF DIETHYLDITHIOCARBAMATE (DDC) AND GLUTATHIONE (GSH) ON CY~OSOLIC SUPEROXIDE DISMUTASE (SOD) ACTIVITY IN ASTROCYTESIN VITRO~

Time post-DDC W

Time post-DDC W

GSH

DDC

DDC, GSH

4 24

100.6t 1.4” 93.3 f 6.6”‘

95.9 * 5.7”’ 104.0k 4.7”’

98.9i 2.5” 97.1 f 17.0-

4 24

Treatment GSH

DDC

DDC, GSH

101 k 2.1”*

532 + 9.21**

190 + 36.6*

112 + 3.3”’

439 ? 63.1**

116 f 12.4”’

‘Results are expressed as percentages of controls, mean f SE. The Cu concentration in the medium was 0.198 + 0.016 ppm. The mean Cu content of control cells was I32 f 4.3 ppm. n = 3-4 per group. Each value for n represents assaysperformed in triplicate. Statistical significance, based on one-way ANOVA and Tukey post hoc analysis, is indicated by *p < 0.05; **p < 0.01: ns = not significant, from controls.

tration. Post-treatment with 10 mM GSH protected the cells from the lethal effects of DDC and restored both GSH-Px activity and Cu content to control levels within 24 hr. The most pronounced ultrastructural alteration in DDC-treated cells was severe mitochondrial damage with loss of internal structure. The morphological lesions observed in these cells are consistent with peroxidative injury and several other investigators have reported DDC damage to the mitochondria as the primary site of insult (Ranek and

TABLE

2

EFFECTSOF DIETHYLDITHIOCARBAMATE (DDC) AND GLUTATHIONE (GSH) ON GLUTATHIONE PEROXIDASE (GSH-Px) ACTIVITY IN ASTROCYTES IN VITRO~ Treatment

Time

post-DDC W)

GSH

4 24

91.4 + 4.3”’ 112 k5.1”’

DDC

DDC, GSH

62.4 k 2.9**

78.7 f 3.4’.

73.1 +- 3.6”

103

f 5.20%

’ Results are expressed as percentages of controls, mean f SE. The mean specificGSH-Px activity for controls was 320 f 28.3

nmol NADPH oxid/min/mg protein. n = 4-7 per group. Each value for n was performed in triplicate. Statistical significance, basedon one-way ANOVA and Tukey post hoc analysis,is indicated by *p < 0.05; **p < 0.01; ns = not significant, from controls.

Treatment

a Resultsare expressedaspercentagesof controls, mean f SE. The mean SOD activityfor controls was 52.5 f 1.9units/mg protein n = 3-4 pergroup. Each valuefor n was performed in triplicate.Statisticalsignificance,basedon one-wayANOVA and Tukey post hoc analysis,is indicated by *p < 0.05; **p < 0.01; ns = not significant,from controls.

Andreassen, 1977; Lin et al., 1979a, 1979b; Trombetta and Adachi, 1985). Although cells treated with GSH after DDC treatment were significantly protected from DDC cytoxicity, some evidence of chemical insult was evident in these cells. These insults consisted of the number of elec-

TABLE

4

EFFECT OF DIETHYLDITHIOCARBAMATE (DDC) AND GLUTATHIONE (GSH) ON REDUCED (GSH) AND OxrDIZED

(GSSG)

GLUTATHIONE

IN ASTROCYTES

IN VITRO

Treatment

Time post-DDC (hr)

GSH

DDC

GSH” 4 24

171 -r-17.9* 130+ 5.3*

93.7 + 7.0”’ 83.4 + 6.9”’

227 rf-16.3** 203 f 27.6**

96.3 k 6.9”’ 123 k9.3””

DDC, GSH 149 + 6.F 114 +

9.6”’

GSSG *

4 24

243 + 61.7** 194 + 22.0**

Note. Statisticalsignificance,basedon one-way ANOVA and Tukey post hoc analysis,is indicated by *p < 0.05: **p < 0.0 1: ns = not significant,from controls. ‘Results are expressedaspercentagesof control, mean t SE. The mean GSH for controls at 4 hr was 32.8 f 2.67 ng GSH/mg protein. The mean control value for GSH at 24 hr was 47.6 ? 3.84 ng GSH/mg protein. n = 3-5 per group. Each valuefor n was performed in triplicate. ‘Results are expressedas percentagesof control, mean ? SE. The mean GSSG for controls at 4 hr was 6.37 + 0.89 1 ng GSSG/ mg protein. The mean control valuefor GSSG at 24 hr was 7.76 ng f 0.99 1 ng GSSG/mg protein. n = 3-5 per group. Each value for n wasperformed in triplicate.

162

TROMBETTA,

TOULON,

tron-dense whorl bodies seen in the cytoplasm. Previous studies have shown that these structures are formed as a result of membrane degradation (Young et al., 1986). Also, it has been shown that electrophilic radicals derived from endogenous or xenobiotic compounds can act as initiators in the chain of events which leads to the formation of these structures. This usually involves the attack of a toxic compound on the polyunsaturated fatty acids in the cellular membranes with consequent peroxidation of membrane lipids and loss of membrane integrity (Halliwell and Gutteridge, 1985). It appears that the addition of GSH postDDC prevented the lethal effects of DDC on cell function or at least stabilized function to the point at which repair could be initiated. The end product of this injury was observed as accumulation of lysosomal degradation products, particularly residual bodies (Fig. 6). Since DDC is a well-known Cu chelator (Cocco et al., 198 1) and since this metal is required for the activity of SOD, several investigators have implicated DDC-induced reduction in SOD activity as the mechanism of toxicity (Lin et al., 1979a, 1979b; Westman and Marklund, 1980; Puglia and Loeb, 1984). Although we were able to document DDC-induced reduction in SOD activity, this occurred only at very high concentrations of DDC-20-fold higher than that required to demonstrate toxicity. At the concentrations used in this study, it is entirely plausible that DDC serves to transport Cu into the cell. It has been reported that DDC binds to Cu to form a highly lipophilic complex which can facilitate the accumulation of this ion within the cell (Johansson and Stankiewicz, 1985). These results call into question the significance of alterations in SOD activity as a mechanism of DDC toxicity. They also support the findings of Mohindru et al. (1983) in which the toxicity of DDC to murine leukemia cells in culture was prevented by the addition of the copper-specific chelator bathocuproine sulfonate. Increased cellular Cu has been shown to enhance lipid peroxidation (Hochstein et al.,

AND

JAMALL

1980; Dougherty and Hoekstra, 1982). Cu can oxidize essential thiol groups, effecting an imbalance in the cellular redox system (Hochstein et al., 1980). The marked increases in GSH and GSSG content above control values in cells treated with GSH alone and in cells treated with DDC and posttreated with GSH (Table 4) may simply reflect increased activity of the GSH-GSSG pathway in the presence of increased substrate (GSH) since the ratio of GSH/GSSG remained unaltered relative to control cells (data not shown). DDC treatment did not affect GSH or GSSG levels, indicating that this substance does not substantially react with GSH or GSSG under these conditions and that the 40% reduction in the activity of GSH-Px is not a reflection DDC-induced reduction in GSH levels per se. Since it is not certain whether exogenous GSH can enter cells (Meister, 1984) the data presented here do not allow for the determination of whether or not the protective effects of exogenous GSH result from interactions with DDC outside or inside the cell. Recent evidence suggests that GSSG is actively transported out of red blood cells (LaBelle et al., 1986a, 1986b). Whatever, the precise mechanism for this interaction, the data clearly demonstrate that Cu plays a key role in the toxicity observed since exogenous GSH preserved the structural integrity of the cell and lowered cellular copper content. Another possibility for cell injury may be the formation of site-specific superoxide anions as a result of the reactivity of cupric ions with the membrane thiols (Hochstein et al., 1980). Superoxide anions, in turn, are capable of generating highly reactive hydroxyl radicals (HO’) in the presence of available hydrogen peroxide and cuprous ions (Hochstein et al., 1980; Halliwell and Gutteridge, 1985). Recent evidence suggests that mitochondrial and cytosolic aldehyde dehydrogenase may play an important role in detoxifying trans-4-hydroxy-2-nonenal and tram-2-hexenal, two cytotoxic products of peroxidized lipids (Mitchell and Petersen, 1987). Since di-

GSH AND DIETHYLDITHIOCARBAMATE

sulfiram is known to inhibit aldehyde dehydrogenase (Sanny and Weiner, 1987) and since our data suggest that the mitochondrion is the Primat?

site of DDC-induced

ultra-

structural injury, the role of aldehyde dehydrogenase in such injury will be examined. Studies are in progress to examine the nature of the DDC-Cu interactions and the protective effects of exogenous GSH in viva REFERENCES AASETH, J., ALEXANDER, J., AND WANNAG, A. (1981). Effect of thiocarbamate derivatives on copper, zinc, and mercury distribution in rats and mice. Arch. Toxicol. 48,29-39. BRADFORD, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem.

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