TOXICOLOGY AND APPLIED PHARMACOLOGY 69, 1 lo- I I6 ( 1983)

Does Consumption of Alcohol in the Presence of Disulfiram Cause Brain Damage?

STEPHEN PHILLIPS’ AND BRIAN CRAGG

Department of Physiology, Monash University, Clayton. Vicroria, 3168, Australia

Received December 4, 1982; accepted February 7. I983

Does Consumption of Alcohol in the Presence of Disulfiram Cause Brain Damage? PHILLIPS, S. C., AND CRACC, B. G. (1983). Toxicol. Appl. Pharmacol. 69, 1 IO- I 16. After blocking aldehyde dehydrogenase activity with disulfiram in rats, a single injection of ethanol or a single day of inhaling ethanol vapor resulted in neuronal degeneration in the cerebral cortex and elsewhere that was detected by a specific silver stain and by electron microscopy. Treatment of rats with alcohol alone and with disulfiram alone did not cause neuronal degeneration. The minimum blood alcohol levels and disulfiram doses administered to the rats which caused neural degen- eration are not distant from those values described in a clinical case of abuse of disulfiram.

Acetaldehyde is the first product of ethanol oxidation and is usually at a low concentra- tion in the blood of healthy subjects drinking alcohol, but higher in alcoholics and their rel- atives. Korsten et al. ( 1975) measured 0.13 mg/dl in controls and up to 0.2 mg/dl in al- coholics whose blood alcohol was above 90 mg/dl. Schuckit and Rayses (1979) obtained the rather higher estimates of 0.2 mg/dl in their controls and 0.3 mg/dl in the relatives of alcoholics. Disulfiram (Antabuse) is still in therapeutic use to block aldehyde dehydro- genase (Woolley and Devenyi, 1980; Gardner and Clarkson, 198 I), and alcohol consump- tion can then lead to high levels of acetalde- hyde (5 mg/dl in rats, Westcott et al., 1980). It does not seem to be known what level of acetaldehyde is sufficient to cause neuronal degeneration and thus contribute to the men- tal deterioration that can be measured in chronic alcoholism (Acker, 1982).

Acetaldehyde has been shown to have greater neuronal toxicity than alcohol when applied to cultures of rat sympathetic gan- glion cells (Eranko et al., 1977) or when ap-

’ To whom requests for reprints should be addressed.

plied topically to rat cerebral cortex (Phillips, 198 1). However, when applied for 1 hr to cor- tex, acetaldehyde at the enormous concentra- tion of 48 mg/dl in the brain tissue did not cause degeneration that could be stained by the Nauta method and its variants, or rec- ognized by electron microscopy, though 130 mg/dl did produce neuronal degeneration (Phillips, 198 1). Thus acetaldehyde is not a fast-acting toxin, and short exposure to a high concentration need not have an irreversible effect. A sufficient duration of exposure must be allowed for acetaldehyde to exert its toxic actions, and a further survival period is needed for neuronal degeneration to become detect- able by histological methods.

We gave rats disulfiram and set out to de- termine how long an exposure to alcohol (and consequently to acetaldehyde) was needed to cause neuronal degeneration. We were sur- prised to find that a single administration of alcohol is sufficient to cause degeneration in the cerebral cortex and elsewhere.

METHODS

Adult male Wistar rats were used throughout this study. Ethanol (cane spirit, CSR) was administered either by

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ALCOHOL, DISULFIRAM. AND BRAIN DAMAGE 111

injection (3 to 4 g/kg ip as a 10% solution in saline) or by vapor inhalation with a vaporizing apparatus described previously (Phihips and Cragg, 1982a). High blood ac- etaldehyde levels were obtained by sc injection of 200 mg disuhiram (Sigma Chemical Co., St. Louis, Mo.)/kg as a 10% solution in olive oil 24 hr prior to ethanol admin- istration and on every second day of ethanol administra- tion.

Blood samples were collected in capped I-ml centrifuge tubes by snipping off the end of the tail while the animal was intoxicated. For ethanol assay, whole blood was de- proteinized in 2% cold trichloracetic acid. Details on the enzymatic assay procedure have been published previ- ously (Phillips rt al.. 1981). For acetaldehyde assay. fresh blood was immediately centrifuged, and the plasma was stored at -20°C for up to 1 week. Without deproteini- zation. samples of 5 ~1 were assayed in a Hewlett-Packard 402 high-efficiency gas chromatograph with a 6-foot length Pyropak Q column. Following the technique of LeBlanc et al. (1975) we used propanol as an internal standard. Blood oxygen saturation levels were measured with a Ra- diometer Hemoximeter OSM 2 to check the possibility that hypoxia might contribute to brain damage.

Animals were killed by an overdose of chloroform va- por, and the anterior half of the body perfused by injec- tion of 60 ml 4% parafotmaldehyde in 0.1 M sodium phosphate buffer, pH 7.4, into the ascending aorta after opening the right auricle. Neural degeneration was stained in frozen parasagittal sections of 30-pm thickness by a modification (Anker and Cragg, 1974) of the Fink and Heimer (1967) method in which silver granules form on membranes of degenerating axons (Heimer, 1970). For electron microscopy. other rats were perfused with 1% glutaraldehyde in phosphate buffer, and small blocks of occipital cerebral cortex were prepared by conventional treatment with osmium tetroxide and uranyl acetate. Thin sections were stained with lead citrate and examined in a Jeol 100s microscope.

As a routine test of the reliability of the stain (Anker and Cragg. 1974). sections from a brain intentionally damaged by applying heat directly to the skull were stained with the sections from the experimental brains.

RESULTS

Blood alcohol. Intraperitoneal injection of 4 g/kg ethanol produced anesthesia in 15 min that lasted for 1 hr, and the rat was able to right itself after 3 hr and stand after 6 hr. The peak blood alcohol was 480 mg/dl and at this stage blood oxygen saturation was 86% (n = 3) which was normal for the tail blood of rats. When ethanol was administered as a vapor for 9 hr, the blood concentration ranged from 190 to 280 mg/dl (n = 3).

Blood acetaldehyde. After injection of di- sulfiram the day before, 3 g/kg ethanol gave acetaldehyde concentrations of0.7 to 0.83 mg/ dl (n = 3) in tail blood 2 hr after alcohol injections. An injection of 4 g/kg ethanol led to 0.6 to 1.3 mg/dl acetaldehyde (n = 3) in tail blood, with oxygen saturation of 8 1%. These values for acetaldehyde are within the range reported by Westcott et al. (1980). It is difficult to compare results with other re- searchers who have obtained their blood sam- ples at different times after alcohol adminis- tration. Inhalation of ethanol vapor resulted in acetaldehyde levels of 0.4 to 0.58 mg/dl, with blood oxygen saturation of 75%.

Brain histology. Six rats were exposed to ethanol vapor alone for 9 hr each day for 3 weeks. Sections stained by our modified Fink- Heimer method showed absolutely no sign of degeneration in any brain region in any of these rats (Fig. I), although concurrently stained sections from a deliberately lesioned brain were strongly impregnated. Sections from an untreated rat brain showed no sign of degeneration in any brain region. An even more severe treatment was given to four other rats who received ethanol vapor 9 hr each day for 3 weeks, followed by I week of withdrawal from ethanol and then 1 week of exposure to ethanol for 24 hr each day. Altogether three cycles of 1 week of withdrawal followed by 1 week of continuous exposure were given. Three of these rats had no trace of axonal degeneration anywhere in the brain, and the fourth had a few sparse and widely separated groups of silver granules in the cerebellar nu- clei, cerebral cortex, and elsewhere. Three ad- ditional rats were given disulfiram alone every second day for 2 weeks without ethanol, and these showed no degeneration in any brain region.

The remaining rats were pretreated with di- sulfiram and then exposed to ethanol, and will be described in descending order of duration of treatment. Six rats were exposed to ethanol vapor for 9 hr each day for 2 weeks, with disulfiram every second day. The brains showed dense impregnation with silver gran-

112 PHILLIPS AND CRAGG

FIG. I. Rat occipital cerebral cortex, layer I, showing absence of degeneration after exposure to ethanol vapor alone for 3 weeks. Bar is 50 pm in Fig. 1 through 4.

ules in all layers of the cerebral cortex (Fig. 2), in the perforant axons to the hippocampus (Fig. 3), in the anterior thalamic nuclei, and in the prepitiform cortex, with sparse silver granules in the cerebellar nuclei and in the glomeruli of the olfactory bulb. Four rats were exposed to ethanol vapor as above for 3 days, and survival for 5 days without ethanol or disulfiram was then allowed for any degen- eration to become manifest. The brains showed strong impregnation with silver gran- ules with the same distribution as above. Six rats were exposed to ethanol vapor as above for only 24 hr and then allowed 6 days of survival, Five of the brains showed definite impregnation with silver granules in layer I of the cerebral cortex (Fig. 4), in the perforant axons in the hippocampus and sparsely in the cerebellar nuclei, with degeneration spreading

to deeper layers of the cerebral cortex in three brains.

Since human exposure to alcohol after treatment with disulfiram would involve one or possibly more drinks rather than inhala- tion of vapor, we treated rats with disulfiram and then a single injection of ethanol (10% w/v in saline, ip). Nine rats were dosed at 4 g ethanol/kg body weight, five died within 24 hr, and the remaining four rats were allowed to survive 6 days without any further treat- ment. All showed fine silver granules in layer I of the cerebral cortex and in the anterior thalamic nuclei: three had silver grains in the olfactory glomeruli, two in the cerebellar nu- clei, and one in the hippocampal perforant tract. After a lower dose of 3 g ethanol/kg body weight, all four treated rats survived 6 days. and all showed silver granules in layer

FIG. 2. The same after treatment with disulfiram and ethanol vapor for 2 weeks,

ALCOHOL. DISULFIRAM, AND BRAIN DAMAGE 113

were also present, but the most numerous ob- jects were enlarged lysosomes, mainly in as- trocytic processes (Fig. 5).

DISCUSSION

Electron microscopy confirms the Fink- Heimer method in showing that exposure to acetaldehyde can cause neuronal degenera- tion, but it is not known whether lysosomes in astrocytes can contribute to the finer silver granules visible to the light microscope (Hei- mer, 1970). We found a sparse distribution of silver granules in the central cerebellar nuclei to which the Purkinje cells project, but counts of Purkinje cells after 2 weeks of exposure to ethanol vapor and disulfiram did not detect a significant loss (Phillips and Cragg, 1982b). In the cerebral cortex, however, especially in

FIG. 3. Degeneration in the perforant axons to the hip- pocampus in a rat given the same treatment as that in Fig. 2.

I of the cerebral cortex, but not in the deeper layers. One of the four also had definite de- generation among the perforant axons in hip- pocampus, and some silver granules in the central cerebellar nuclei.

As shown in Figs. 2 to 4, many of the silver granules were very fine, and there were rather few of the rows of dashes that usually mark degenerating axons. This finding suggested that acetaldehyde might be causing a dying- back process at axon terminals. We therefore examined cerebral cortex of rats exposed to disulfiram and ethanol vapor by electron mi- croscopy. After 7 to 9 days of ethanol treat- ment, many degenerating axon terminals were seen in the cerebral cortex (Fig. 5), but we were not able to determine how far along the axons the blackening extended. A few degen- FE. 4. Cortical layer I 6 days after treating the rat with erating cell bodies (Fig. 6) and axons (Fig. 7) disulfiram and ethanol vapor for 24 hr.

114 PHILLIPS AND CRAGG

FIG. 5. Rat cerebral cortex after 7 days treatment with disulliram and ethanol vapor. showing degenerating axon terminals (stars) and lysosomes (arrows). Bar is I Frn in Figs. 5 through 7.

layer I (Figs. 2 and 5) there was a substantial amount of neuronal degeneration. We had previously found that exposure to 48 mg/dl acetaldehyde in cerebral cortex for 1 hour did not cause any neuronal degeneration in the treated cortex that was visible to light or elec- tron microscopy (Phillips, 198 1). In the pres- ent experiments 0.4 to 0.6 mg/dl acetaldehyde in blood for 24 hr when ethanol vapor was administered did cause degeneration. Thus some step requiring time is needed to initiate cell dying at these concentrations, and this step might possibly be the interference by ac- etaldehyde with enzymic reactions that are involved directly or indirectly in protein syn- thesis (Cederbaum and Rubin, 1977; Lindros, 1978). Our gas chromatograms did not detect acetone in the blood samples, although an el- evated level of acetone has been found by oth- ers after disulfiram treatment (De Master and

Nagasawa, 1977). In spite of the neuronal de- generation after disulfiram and exposure to alcohol vapor for 2 weeks, we did not detect any weakening of the blood-brain barrier as measured with radioactive sucrose (Phillips and Cragg, 1982a).

It is not clear how often humans with al- coholism would experience a toxic level of acetaldehyde. Veghelyi et al. (1978) claim to have observed acetaldehyde levels as high as 1 mM (44 mg/dl) in some alcoholic patients receiving disulfiram treatment, but this amount is greatly in excess of the measure- ments in man and rats quoted in the Intro- duction. Deproteinization of blood in the presence of ethanol can lead to the production of acetaldehyde (Stowell et al., 1977) thus giv- ing a misleadingly high concentration. The artifactual formation of acetaldehyde is avoidable by immediate centrifugation of the

ALCOHOL, DISULFIRAM, AND BRAIN DAMAGE 115

fresh blood to remove the red blood cells from the biological sample (Eriksson, 1980). An in- toxicated heavy drinker who ingested 7 g of disulfiram tablets and continued to drink thereafter was reported by Woolley and De- venyi (1980). Acetaldehyde was not mea- sured, but blood alcohol was 178 mg/dl on admission to hospital 14 to 19 hr after taking disulfiram. We have produced cortical degen- eration in five of six rats treated with disul- firam and ethanol vapor to a blood ethanol level of 190 to 280 mg/dl. There is thus a considerable risk of causing brain damage by abuse of disulfiram therapy.

Moreover, acetaldehyde in rats caused neu- ronal degeneration at 0.7 mg/dl after a single injection of alcohol, and at 0.4 mg/dl after inhalation of ethanol vapor for 24 hr. Acet- aldehyde levels up to 0.2 mg/dl have been reported in some human alcoholics not using disulfiram (Korsten et al., 1975). We do not yet know how low an acetaldehyde level can

FIG. 7. Degenerating myelinated axon after same treat- ment as in Fig. 5.

be when sustained over a long period and still be effective at causing brain damage.

ACKNOWLEDGMENTS

This research was funded by Monash University and the Australian Associated Brewers.

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