Proc. Natl. Acad. Sci. USA Vol. 76, No. 10, pp. 5303-5307, October 1979 Medical Sciences

Tetraethylthiuram disulfide (Antabuse) inhibits the human malaria parasite Plasmodium falciparum (malaria/in vitro culture/disulfiram) L. W. SCHEIBEL, A. ADLER, AND W. TRAGER Laboratory of Parasitology, The Rockefeller University, New York, New York 10021 Contributed by William Trager, May 30, 1979

ABSTRACT Plasmodium falciparum in culture grows op- effects apart from those following ingestion of alcohol. timally at 3% oxygen. Oxygen levels down to 0.5% still support Therefore it has received attention in aversion therapy in the growth, but anaerobic conditions do not. These findings, and the absence of the Krebs cycle in Plasmodium, suggested that treatment of alcoholics (12), as well as heavy metal poisoning in this organism oxygen may not function in electron transport (7). Second, these compounds exhibit lipid/H20 partition but rather may act through metalloprotein oxygenases. coefficients favoring penetration (7). This is an important Tetraethylthiuram disulfide (Antabuse, disulfiram) and its re- consideration because the compound must penetrate the duction product diethyldithiocarbamate inhibit many metal- erythrocyte membrane, equilibrate with the erythrocyte loprotein oxygenases and have a lipid/H20 partition coefficient cytoplasm, then traverse the parasitophorous vacuole mem- and high binding constant for metal ions, favoring selective toxicity to the malaria parasite. These compounds exhibited brane and space, and finally penetrate the limiting membranes active antimalarial effects in vitro in concentrations down to of the intracellular parasite and possibly even subcellular or- 0.1 ,g/ml, the lowest level tested. Tetraethylthiuram disulfide ganelles. Increasing the hydrocarbon chain of the alkyl groups at a level as low as 1 pg/ml inhibited parasite glycolysis with or introducing aryl groups increases the lipophilic characteristics no effect on glycolysis of normal erythrocytes. Erythrocytes of the drug molecule but unfortunately decreases activity, pretreated with this drug at 10 pg/ml did not support growth probably due to steric hindrance (9). Last, these compounds of the parasite. have favorable binding constants enabling them to compete Plasmodium falciparum, when cultured in vitro (1), has been effectively with naturally occurring chelators in the cell for shown to be a microaerophile, growing best at 3% 02 but tol- metal ions (13-15). Therefore, on the basis of these favorable erating 02 levels as low as 0.5% without appreciable reduction parameters one would expect DDC and its dimeric oxidation in multiplication rates (2). However, there does appear to be product tetraethylthiuram disulfide (TETD) to exhibit highly a critical level of 02 below which the parasite will not survive, selective inhibitory activity toward the malaria parasite P. in contrast to true anaerobic forms of life. It has been known falciparum. for some time that the malaria parasite does not use the Krebs cycle to degrade its glucose completely to CO2 and H20 but MATERIALS AND METHODS instead excretes organic end products (3, 4). This suggests that P. falciparum, strain FCR3/FMG (Gambian strain) (16) was the parasite may depend on reactions involving oxygenases and grown in vitro in 8% erythrocyte suspensions by using the petri hydroxylases in which 02 is a chemical reactant (5). These ox- dish-candle jar method as described by Jensen and Trager (17). idations are catalyzed by oxidase enzymes or enzyme systems Type A erythrocytes and sera were used throughout the study. that activate the 02 and oxidize the substrate. Oxidases are often Four petri dishes of parasites were grown at each drug con- metalloproteins, containing metals such as iron, copper, or centration unless otherwise specified. The media and parasit- molybdenum, and are present in microorganisms, plants, and ized erythrocytes were dispensed into petri dishes and parasites animals (6). Therefore, we felt, first, that a chelating agent with were allowed to multiply in the absence of drug for 24 hr. activity against such metalloproteins might have selective Smears were made of all cultures daily and stained with toxicity to malaria parasites without interference with the Giemsa's stain. A minimum of 1000 erythrocytes were counted cellular metabolism of the host tissues. One type of agent on each slide and growth was expressed each day in percentage emerges from the rest with high probability of achieving this erythrocytes infected with P. falciparum. Initial parasitemia objective, the dialkyldithiocarbamates and their dimers, the was determined by counting parasites in 40,000 erythrocytes. thiuram disulfides. Diethyldithiocarbamate (DDC) is a che- TETD was dissolved in dimethyl sulfoxide (Me2SO), certified lating agent with proven inhibitory activity against many of Spectranalyzed (Fisher), to give a concentration of 80 mg/ml. these enzymes (7). There is no sharp distinction among the alkyl Fifty microliters of this solution was added to 40 ml of medium. dithiocarbamoyl compounds, and they can be transformed into Controls contained equal quantities of Me2SO only. DDC, so- each other in nutrient solutions (8). Dimethyldithiocarbamate dium salt, was dissolved directly in 40 ml of medium. Stock is strongly fungotoxic, particularly to fungi with obligatory solutions were sterilized by filtration and appropriately diluted aerobic metabolism. Fortunately, higher plants and vertebrate with sterile medium to achieve desired concentrations of drug. animals possessing an obligatory aerobic type of metabolism TETD and DDC were recrystallized reagents. show little or no response to these substances (7, 9, 10). The di- For the determination of glucose, lactate, pyruvate, and ethyl derivative (Antabuse, disulfiram) is even lesis toxic to protein, perchloric acid was added to the cultures to a final mammals (11), and, in clinical trials, single doses of up to 6 g concentration of 3%. The protein was removed by centrifuga- and daily doses of 0.25-0.60 g for several months produced no tion at 27,000 X g for 15 min and was determined in the pellet by the procedure of Lowry et al. (18). The supernatant was The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- Abbreviations: TETD, tetraethylthiuram disulfide (Antabuse or di- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate sulfiram); DDC, diethyldithiocarbamate; Me2SO, dimethyl sulf- this fact. oxide. 5303 Downloaded by guest on September 29, 2021 5304 Medical Sciences: Scheibel et al. Proc. Nati. Acad. Sci. USA 76 (1979)

Table 1. Growth of P. falciparum in DDC DDC, % parasitemia* ,ug/ml 2 days 3 days 4 days 5 days 100 0 0 0 0 10 0 0 0 0 1 0.6 (0.4-0.8) 0.5 (0.4-0.6) 0.5t (0.4-0.7) 0.5t (0.3-0.7) 0.1 0.8 (0.5-1.0) 1.4 (0.8-1.8) 1.2 (0.9-1.7) 0.4t (0.1-0.8) 0 1.1 (1.0-1.3) 1.5 (1.2-1.9) 6.5 (4.1-8.0) 8.0 (6.3-9.6) Parasites were grown 24 hr in 1.5-ml petri dishes in a candle jar before exposure to DDC. * % parasitemia is expressed as infected erythrocytes per 100 erythrocytes. The initial parasitemia was 0.75%. The average and range (in parentheses) are given from counts on four dishes. This is the result of one of three similar experiments. t Grossly abnormal or dead.

neutralized with KOH. Lactate was quantified enzymatically This suggests an immediate effect on the development of ma- by. a modification of the method of Lowry et al. (19). Lactate laria by DDC or TETD. Concentrations as low as 0. l ,ug/ml dehydrogenase (Worthington) used in this assay was diluted exhibited an 82% reduction in growth 72 hr after the addition with 0.9% NaCl rather than albumin and the buffer used was of DDC to the culture (96 hr after the start of the experiment) 2-amino-4-methyl-1-propanol, pH 9.7. Pyruvate was deter- and no survivors at the completion of the experiment. Similar mined according to the method of Bucher et al. (20) except for results were seen with TETD: a 50% reduction in growth 72 hr the substitution of 0.5 M glycylglycine buffer in place of 0.4 M after administration of the drug and a 74% reduction at the end triethanolamine buffer. Glucose was assayed spectrophoto- of the experiment. Thus both TETD and its physiological re- metrically at-340 nm by a modified method of Slein (21). The duction product, DDC, showed an equivalent effect. This in- reaction mixture contained 40 ,gmol of glycylglycine buffer (pH hibition appeared to be directly proportional to the concen- 7.4), 0.8 limol of MgCI2, 3.75 jAmol of ATP and 0.3 ,umol of tration of the compound used. NADP+, 0.56 unit of hexokinase and 0.28 unit of glucose-6- Effect of TETD on Glycolysis of P. falciparum-Infected phosphate dehydrogenase (Boehringer and Soehne) in 0.05 M and Normal Erythrocytes. Evidence has been reported that glycylglycine buffer (pH 7.4). The total volume was 1.0 ml. these compounds exhibit their toxic effects on sensitive fungi Studies involving "pretreated" erythrocytes utilized unin- by inhibition of the paired mercapto groups of the lipoic fected erythrocytes stored in the refrigerator in TETD at a total acid/dehydrogenase system (23). Therefore, assessment of concentration of 10 ,ug/ml for 24 hr. These were then washed glycolysis by measuring glucose utilization and lactate/pyruvate three times in a 2-fold excess of drug-free medium and mixed production in parasitized and normal erythrocytes was done with concentrated samples of predominantly schizonts sepa- in TETD and Me2SO controls (Table 3). Examination of slides rated in gelatin (22) at time zero. This mixture was added to made at the start of incubation with the drug and 22 hr later, 3-cm diameter petri dishes in normal medium to make a final at termination, showed no change in parasite numbers or 8% cell suspension and incubated in a candle jar at 380C. Daily morphology except that the 100 Ag/ml dose appeared to kill changes were done with drug-free complete medium and slides the plasmodia. In conformity with this finding, there was 100% were made to assess parasitemia. inhibition of glucose utilization and lactate production over the last 16 hr at this high drug dose. RESULTS With TETD at 10 ,gg/ml, there was only a 44% inhibition of Growth at Different Concentrations of DDC and TETD. glucose utilization, significantly less than the 70% inhibition Typical effects of growth of P. falciparum in DDC or its oxi- achieved with TETD at 1 ,g/ml. This is termed "concentration dation product TETD at concentrations of 100-0.1 ,ug/ml are quenching" or "biphasic response" (15) and in general agrees shown in Tables 1 and 2. It can be seen that there was no mul- with the results reported by others on pyruvate production in tiplication of the parasites as early as 24 hr after the addition fungi in the presence of these compounds (24, 25). This is de- of concentrations of these compounds greater than 1 jig/ml. termined by the type of complex formed between chelator and Table 2. Growth of P. falciparum in TETD TETD,* % parasitemiat ,Ug/ml 2 days 3 days 4 days 5 days 100 0.2 (0.1-0.2) 0 0 0 0 0.7 (0.4-1.0) 1.4 (1.1-1.6) 5.8 (5.4-6.8) 9.7 (8.2-11.1) 10 0.2 (0.2-0.2) 0.2 (0.1-0.2) 0 0 0 1.2 (0.9-1.4) 1.6 (1.3-1.9) 5.5 (4.4-7.0) 7.5 (6.5-8.0) 1 0.8 (0.6-1.1) 0.6 (0.3-0.9) 0.5t (0.4-0.7) 0.3t (0.2-0.6) 0 1.0 (0.8-1.2) 1.5 (1.2-1.8) 6.5 (4.8-7.4) 9.5 (8.2-10.8) 0.1 1.2 (0.8-1.5) 1.3 (0.8-2.1) 3.2 (2.5-4.5) 2.4 (1.5-3.2) 0 1.0 (0.7-1.5) 1.4 (1.2-1.6) 6.2 (4.9-7.1) 9.1 (8.0-11.4) Parasites were grown 24 hr if 1.5-ml petri dishes in a candle jar before exposure to TETD. * In all cases 0 indicates the Me2SO control. t % parasitemia is expressed as infected erythrocytes per 100 erythrocytes. The initial parasitemia was 0.75%. The average and range (in parentheses) are given from counts on four dishes. This is the result of one of six similar experiments. Grossly abnormal or dead. Downloaded by guest on September 29, 2021 Medical Sciences: Scheibel et al. Proc. Natl. Acad. Sci. USA 76 (1979) 5305

metal ion. DDC can form two complexes with divalent cations It is significant, however, that TETD at 100, 10, or 1 ,g/ml [metal.DDC and metal.(DDC)2]. Presumably, the 1:1 complex had no effect on glycolysis of the uninfected erythrocytes. This predominates at low drug levels and is maximally inhibitory suggests that inhibition of glycolysis by TETD is specific for to glycolysis. This is termed "first zone of inhibition." Higher parasitized erythrocytes even though both the malaria parasite drug concentrations favor formation of the 1:2 complex, which and the mature erythrocytes rely on glycolysis for their me- is less inhibitory (7). As the concentration of drug is raised still tabolism. It is also significant to note there is a marked effect further, free dithiocarbamate predominates, resulting in a re- on glycolysis in the infected cells at doses 1/10th those reported bound effect, termed a "second zone of inhibition" (24, 26). It as normal serum levels (27, 28). is interesting to note that this concentration quenching effect Growth of P. falciparum in Sera from Volunteers on was not seen by us in the growth studies of P. falciparum (Ta- TETD. In an effort to test whether, in fact, the effect is on re- bles 1 and 2), suggesting that growth inhibition by these com- cipient erythrocytes, two volunteers with type A blood were pounds in malaria may be the result of another biochemical placed on therapeutic doses of TETD (500 mg orally once a mechanism. It is known that EDTA is able to compete effec- day) for 4 days. Blood was drawn from them 4 hr and 4 days tively with DDC for divalent cations extracellularly, reducing after the start of the experiment. The serum was separated from toxicity at low concentrations of the thiocarbamate (26). In our the erythrocytes and frozen for use in the in vitro cultivation studies, however, 0.5 mM EDTA had no antagonistic effect on system. This in vitro system for growing P. falciparum nor- growth inhibition by DDC or TETD. This further indicates mally employs only a 10% concentration of serum in RPMI growth inhibition by these compounds may not be related to 1640 medium (17). To approach the pharmacologic levels of the decrease in glycolysis in TETD. In contrast, the inhibition drug circulating in the volunteers, it was necessary to raise the of glucose utilization by TETD in the first 6 hr was low. The proportion of serum used in the test system. Our studies indi- effect at doses of 100 and 1 jug/ml was almost insignificant cated that normal serum could be added to the in vitro culti- compared to the effect seen at 10 ug/ml. Replicate experiments vation system as high as 50% without deleterious effects on the at 6 hr resulted in similar low utilizations with small quantitative growth of the parasite, provided the final glucose concentration variations. This suggests that the primary inhibitory effect of was 2 mg/ml. TETD on P. falcdparum may not be through glucose utilization. Sera from volunteers taking TETD were then added to the Similar findings were reported in fungus cultures treated with culture system at a 50% concentration and supplemented with the closely related compound dimethyldithiocarbamate (25). glucose (2 mg/ml). This serum did not inhibit the growth of P. On the other hand, the biphasic response is seen in lactate falciparum, suggesting either that the drug is partitioned production at both 6 hr and after an additional 16 hr of incu- rapidly in the erythrocytes in vivo and actually is not in the bation. Both 100 and 1 Ag/ml concentrations of TETD gave serum, or that it is bound too tightly to pass into recipient greater inhibition than the 10.ug/ml dose. The effect on lactate erythrocytes to be inhibitory to the parasite. production in the first 6 hr of incubation paralleled the 16-hr P. falciparum Infections of Erythrocytes Treated with results. In contrast to data reported on fungi, pyruvate did not TETD. Linderholm and Berg (29) reported that, after ad- accumulate to any significant extent in our experiments on P. ministration of TETD to humans, appreciable amounts of DDC falciparum. Levels of pyruvate were low, none being detected can be found in erythrocytes as long as 30 hr after ingestion of in 6 hr and only 0.01-0.02 ,mol/mg of protein accumulating the drug. Therefore, erythrocytes were pretreated with TETD in 16 hr. The concentration quenching results were also evident at 10 Ag/ml (equivalent to serum levels reported in humans here and reflect those seen in lactate production. No pyruvate taking therapeutic doses of this drug) for 24 hr in the refriger- accumulated in uninfected cells. ator and then thoroughly washed. Such cells did not support Table 3. Effects of TETD on glucose utilization and the formation of lactate in normal and infected erythrocytes (RBCs) by P. falciparum Glucose utilized Lactate produced Infected RBCs Normal RBCs Infected RBCs Normal RBCs Incubation, TETD,* Omol/ % gmol/ Omol/ % Omol/ hr Ag/ml mg protein inhibition mg protein mg protein inhibition mg protein 6 100 0.268 6.9 0.062 0.021 94 0.024 0 0.288 0.077 0.342 0.027 10 0.259 27.7 0.083 0.302 8.9 0.024 0 0.358 0.087 0.332 0.027 1 0.328 0 0.072 0.261 25.5 0.033 0 0.318 0.057 0.350 0.028 Additional 16 100 0 100 0.058 0 0.084 0 0.573 0.032 1.476 100 0.113 10 0.342 43.9 0.070 1.151 0.134 0 0.610 0.049 1.398 0.118 1 0.207 70 0.066 0.886 0.129 0 0.608 0.064 1.429 38.7 0.116 Parasites were grown in.4-ml petri dishes to a 7% parasitemia. Slides were made to assess parasitemia. TETD was added in specified concentrations, each accompanied by a Me2SO control. Zero time dishes were immediately harvested. Growth in the remaining dishes was allowed to continue for 6 hr, and the supernatants were removed for analysis. Fresh media containing TETD or Me2SO were added and parasites were allowed to grow an additional 16 hr. Slides were made for all petri dishes to assess growth and morphology. All reactions were stopped by the addition of perchloric acid to a final con- centration of 3%. This is the result of one of two similar experiments. * Controls contained the same amount of Me2SO as was introduced with the stated concentration of TETD. Downloaded by guest on September 29, 2021 5306 Medical Sciences: Scheibel et al. Proc. Nati. Acad. Sci. USA 76 (1979) Table 4. Effect of pretreatment of erythrocytes with TETD on subsequent growth of P. falciparum % parasitemia Pretreatment 0 days 1 day 2 days 3 days 4 days 5 days 6 days Normal medium 0.7 0.4 (0.3-0.4) 0.6 (0.4-0.8) 1.6 (1.0-2.4) 4.6 (3.2-5.7) 5.9 (3.6-6.9) 15.2 (12.6-18.7) Normal medium + Me2SO 1.0 0.4 (0.3-0.5) 0.6 (0.4-0.8) 1.7 (1.3-1.9) 5.7 (5.4-6.1) 4.7 (3.8-5.8) 16.6 (15.5-17.6) TETD 0.6 0.4 (0.4-0.5) 0.7 (0.5-0.7) 0.6 (0.5-0.7) 0.3 (0.1-0.4) 0 0 TETD + serum 0.5 0.3 (0.3-0.4) 0.4 (0.3-0.5) 0.4 (0.3-0.4) 0.2 (0.2-0.2) 0 0 This is the result of one of three similar experiments. The hverage and range (in parentheses) are given from counts on four dishes. growth of P. falciparum (Table 4). This suggests the lipophilic It is known that many metalloprotein oxidases in addition to drug may accumulate on or in the erythrocytes, preventing cytochrome oxidase are inactivated by cyanide (42). In contrast, them from supporting growth of the malaria parasite. When the dithiocarbamates show no effect on the cytochrome system the experiment was done with erythrocytes incubated in TETD (43) and affect 02 consumption only in concentrations that far for 24 hr at 380C and subsequently treated as were those pre- exceed those that inhibit growth of organisms in test systems treated in the refrigerator, the inhibition was less dramatic. This (8). Therefore the inhibition of the growth of malaria parasites apparent discrepancy is not yet explained. observed in this study is not likely due to inhibition of terminal electron transport reactions in this organism. DISCUSSION In contrast to the potent in vitro activity of this drug in our TETD is a relatively safe compound used clinically to treat studies, serum drawn from volunteers on therapeutic doses of alcohol abuse patients by aversion therapy (30). It is thought TETD was not inhibitory to P. falciparum in culture at a 50% that this drug sensitizes them to alcohol presumably through serum/medium mixture. Drug concentrations reported to be inhibition of aldehyde oxidase. Failure to abstain from ethanolic circulating in patients taking TETD fall within the "parasiti- beverages results in a rise of serum acetaldehyde and a wide cidal" limits seen in our growth studies. It has been reported that variety of unpleasant systemic manifestations (31), which en- the therapeutic dose of 250-500 mg per day in humans results courages abstinence from spirits. It is interesting to note that in a "plasma" concentration of 0.5-1.0 mg/100 ml, and the inordinately high doses (500-1000 mg/kg) DDC injected in- drug persists in pharmacologically active levels for about 6 days travenously have been reported to damage islets of Langerhans (28). Because TETD in concentrations as little as 1:1,000,000 in experimental animals, thereby causing diabetes (32), prob- (10-7 M) in vitro totally inhibits acetaldehyde oxidase of rabbit ably by reacting with the Zn2+ in the islet cells. However, doses and rat liver (44, 45), the unusually high plasma concentration of sodium DDC required to produce hyperglycemia were of the drug reported must be reexamined. Our studies suggest 500-1000 mg/kg, and the hyperglycemia was only temporary that human erythrocytes treated with levels of TETD reported in 11 out of 15 animals. The lethal dose of TETD is 3 g/kg body as circulating in patients taking this drug did not support growth weight (12, 33), but lower doses are relatively nontoxic apart of parasites as long as 6 days later. This indicates that the from those symptoms seen after ingestion of alcohol. erythrocytes are changed in some way by TETD, maldng them Our studies demonstrate that low doses of TETD and its re- inhospitable to the parasites. It remains to be seen if erythrocytes duction product, DDC, are highly inhibitory to growth of the taken from patients receiving the drug would also have resisted human malaria parasite P. falciparum. This is consistent with infection, because the work of Linderholm and Berg (29) the finding that the closely related compound tetramethylthi- suggests that all of the DDC is found in erythrocytes of humans uram disulfide and its carbamate reduction product are more taking TETD. toxic to parasitic molds than to saprophytic fungi (10). In ad- We thank Dr. S. Sassa for suggestions made during the preparation dition, the glycolysis of the malaria parasite appears specifically of this manuscript and Drs. E. Bueding and J. Bennett for supplying sensitive to low doses of TETD, in contrast to glycolysis in the the recrystallized TETD and DDC used in this study. This investigation host erythrocyte. This may result from differential sensitivity was supported by a contract from the Agency for International De- of host and parasite glycolytic enzymes, or less directly as a velopment, U.S. Department of State. consequence of higher than usual methemoglobin concentra- tion, which is known to be present in malarial erythrocytes (34). 1. Trager, W. & Jensen, J. B. (1976) Science 193,673-675. TETD is rapidly and almost quantitatively reduced to DDC 2. Scheibel, L. W., Ashton, S. & Trager, W. (1979) Exp. Parasitol. (35-37), presumably through the glutathione/glutathione re- 47,410-418. ductase system of erythrocytes (37, 38), in many in vitro systems 3. Scheibel, L. W. & Miller, J. (1969) Mil. Med., Special Issue, 134, and in vivo. The reduction can be demonstrated spectropho- 1074-1080. 4. Scheibel, L. W. & Pflaum, W. K. (1970) Comp. Biochem. Physiol. tometrically at 450 nm (26, 39) and occurs in our in vitro cul- 37,543-553. tivation system, indicating that the chemical moiety DDC is 5. Fairbairn, D. (1970) Biol. Rev. Cambridge Philos. Soc. 45, in fact generated in our system. It is known that methemoglobin 29-72. promotes oxidation of DDC to TETD (35, 36) and TETD but 6. West, E. S. & Todd, W. R. (1961) Textbook of Biochemistry not DDC is a potent inhibitor of hexokinase (35). Therefore, (MacMillan, New York), 3rd Ed., p. 855. cells high in methemoglobin such as the P. falclparum-infected 7. Thorn, G. D. & Ludwig, R. A. (1962) The Dithiocarbamates and cells in culture would be sensitive to TETD inhibition of gly- Related Compounds (Elsevier, New York). colysis, presumably at hexokinase. In any case, it seems likely 8. Goksoyr, J. (1955) Physiol. Plantarum 8,719-835. that the growth inhibition exerted by DDC and TETD does not 9. Klopping, H. L. & Van der Kerk, G. J. M. (1951) Rec. Trav. Chim. stem from its on Pays-Bas 70,917-939. mainly effect glycolysis. 10. Manten, A., Klopping, H. L. & Van der Kerk, G. J. M. (1950) Malaria parasites free from host cell contamination have been Antonie van Leeuwenhoek J. Microbiol. Serol. 16, 45-55. shown to be rich in cytochrome oxidase, a metalloprotein (3, 11. Brieger, H. & Hodes, W. A. (1949) Proceedings, 9th Interna- 40, 41). Our unpublished studies also show that P. falciparum tIonal Congress of Industrial Medicine, London, 1948 (J. is extremely sensitive to KCN at concentrations to 5 ,uM in vitro. Wright, London), pp. 598-602. Downloaded by guest on September 29, 2021 Medical Sciences: Scheibel et al. Proc. Natl. Acad. Sci. USA 76 (1979k 5307

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