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Agents and Actions vol. 5/2 (1975) Birkh/iuser Verlag, Basel
Protective Action of Ascorbic Acid and Sulfur Compounds against Acetaldehyde Toxicity: Implications in Alcoholism and Smoking 1) by HERBERT SPRINCE2), CLARENCE M. PARKER, GEORGE G. SMITH and LEON J. GONZALES U.S. Veterans Administration Hospital, Coatesville, Pennsylvania 19320, and The Departments of Psychiatry and Human Behavior and Pharmacology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA
Abstract Acetaldehyde is a toxic substance common to heavy drinking of alcohol and heavy smoking of cigarettes. It has been implicated thereby in diseases of the cardiovascular, respiratory, and central nervous systems. Protection against acetaldehyde toxicity (i.e. anesthesia and lethality) was studied in rats by oral intubation of test compounds 30-45 minutes prior to oral intubation of a standardized oral LD 90 dose (18 millimoles/kilogram) of acetaldehyde. Animals were monitored for anesthesia (loss of righting reflexes) and lethality for 72 hours. A total of 18 compounds was tested. L-ascorbic acid at 2 miRimoles/kilogram (mM/kg) showed moderate protection against anesthesia and marked protection against lethality. Greatest protection against anesthesia and lethality was obtained at 2 mM/kg with each of the following: L-eysteine, N-acetyI-L-cysteine, thiamin" HCI, sodium metabisulfite, and L-eysteie acid. A combination of L-aseorbic acid with L-cysteine, and thiamin.HCl at reduced dose levels (2.0, 1.0 and 0.3 mM/kg, respectively) gave virtually complete protection. A detailed literature review is presented of the rationale and significance of these findings. Our findings could point the way to a possible build-up of natural protection against the chronic body insult of acetaldehyde arising from heavy drinking of alcohol and heavy smoking of cigarettes.
Preceding publications from this laboratory [1-3] directed attention to a possible means of protection against the adverse effects of acetaldehyde, a key intermediary metabolite of ethyl alcohol (ethanol), more toxic than ethanol, and also a toxic component of cigarette smoke. Currently, the chronic adverse effects of acetaldehyde on body tissues assume even greater significance with the increasing realization that, ~) Supported by U.S. Veterans Administration (Project No. 8078-01 and 8078-04). ,z) Address reprint requests to Dr. Herbert Sprince, VA Hospital, Coatesville, Pennsylvania 19320, USA.
despite its rapid metabolism in vivo, body levels of acetaldehyde may be elevated by both heavy alcohol drinking [4-6] and heavy cigarette smoking [4, 7-9]. This possible relationship of acetaldehyde to the adverse effects of both alcohol and cigarette smoking was clearly stated in the 1970 review of T. N. JAMES et al. [4], and is strengthened by recent reports establishing that heavy drinkers are generally also heavy smokers [10, 11 ]. In the case of alcoholism, acetaldehyde has been implicated in alcoholic cardiomyopathy [4, 12-14], in alcohol addiction [15, 16], in ethnic sensitivity to alcohol [17], in alcoholic cerebellar degeneration [18], in the aging process [19], and in the disulfiram-ethanol reaction [20] used in the aversion treatment of alcoholism. Medically, this last named reaction is of greater importance than generally realized since a disulfiram-like interaction with ethanol also occurs with a number of therapeutic drugs other than disulfiram [21]. Acetaldehyde could also play a role in the development of 'alcoholic lung disease' [22-24]. In the case of heaw cigarette smoking, acetaldehyde has been implicated not only in cardiac disease [12-14] but also in pulmonary disease by way of its highly toxic action on cilia of the respiratory tract [25], an effect believed to be related to the premalignant state of the tracheobronchial tree [26, 27]. More recently, its content in cigarette smoke in association with cigarette tar has been found to correlate highly with the 'free lung cell response' of RYLANDER [8]. This response is defined as the change in numbers of macrophages and leucocytes induced by cigarette smoke and 'probably represents a significant exposure reaction in terms of development of pulmonary disease' (quotation from RYLANDER [8]). From
Protective Action of Ascorbic Acid and Sulfur Compounds
the f o r e g o i n g , it is e v i d e n t t h a t a c e t a l d e h y d e is a toxic s u b s t a n c e c o m m o n to h e a v y a l c o h o l d r i n k i n g a n d h e a v y cigarette s m o k i n g a n d i m p l i c a t e d i n c a r d i o v a s c u l a r a n d p u l m o n a r y disease [4]. T h e r e f o r e it b e c o m e s i m p o r t a n t to s e a r c h for i n vivo p r o t e c t a n t s a g a i n s t its a d v e r s e effects. S u c h protectants (particularly, naturally-occurring ones) c o u l d be o f p o t e n t i a l t h e r a p e u t i c v a l u e as p r e v e n t i v e s for tissues c h r o n i c a l l y e x p o s e d to elevated a c e t a l d e h y d e levels. I n t h e p r e v i o u s p u b l i c a t i o n s f r o m this l a b o r a t o r y [1-3], details were p r e s e n t e d o f t h e protective a c t i o n i n rats o f L-cysteine free base (FB), t h i a m i n - H C I , a n d L - 2 - m e t h y l t h i a z o l i d i n e 4 - c a r b o x y l i c acid a g a i n s t a c e t a l d e h y d e toxicity a n d lethality at a carefully s t a n d a r d i z e d L D 90 dose level. T h i s s t u d y p r e s e n t s o u r f i n d i n g s o n the protective a c t i o n o f r e l a t e d s u l f u r c o m p o u n d s a n d a s c o r b i c acid a g a i n s t a c e t a l d e h y d e toxicity in rats at the s a m e s t a n d a r d i z e d L D 90 dose level.
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Materials and methods
The following is a brief outline of the methodology used; details are set forth in our previous publication [1]. Carworth Farms (CFE) strain male albino rats 90 4- 5 days old and weighing 365 4- 20 grams were deprived of food, but not water, overnight. Next morning, they were reweighed just prior to test for calculation of dosages of the test compounds. Compounds tested and the doses used are listed in Tables 1 and 2 under 'results'. Test compounds were freshly prepared as solutions or fine suspensions in physiological (0.95/00)saline purged with nitrogen gas to protect against undesirable oxidation. Dilutions were made to concentrations convenient for oral intubation of the desired dose at convenient volumes (1-2.5 ml). Acetaldehyde was always freshly distilled just before use. For initial screening, 30 rats per day were generally tested in equal groups (5 rats/group). Such tests were made in duplicate on two consecutive days to give a minimum of 10 rats/group tested. When a given test compound showed definite protection, these tests were replicated with more rats to the total number indicated in Table 1. Test compounds or combinations thereof were intubated orally 30-45 minutes prior to oral intubation of
Table 1
Protective action of L-ascorbic acid and sulfur compounds against anesthetic and lethal effects of acetaldehyde in the rat. (Toxic oral dose of acetaldehyde used was LD 90 = 18 mM/kg.) Compounds tested (oral dose)
Dose (mM/kg)
~ anesthetized after 3-10 minutes
~ dead after 3 hours
24 hours
72 hours
Control (saline)
-
96 (48/50)
54 (27/50)
90 (45/50)
90 (45/50)
2.0 2.0 2.0 2.0 2.0
53 (8/15) 20 (5/25) 60 (6/10) 47 (7/15) 10 (2/20)
27 (4/15) 8 (2/25) 50 (5/10) 0 (0/15) 0 (0/20)
27 (4/15) 20 (5/25) 60 (6/10) 33 (5/15) 0 (0/20)
27 (4/15) 20 (5/25) 60 (6/10) 33 (5/15) 0 (0/20)
2.0 2.0 2.0 2.0 2.0
80 (8/10) 87 (13/15) 90 (9/10) 80 (8/10) 60 (6/10)
20 (2/10) 47 (7/15) 30 (3/10) 10 (1/10) 60 (6/10)
70 (7/10) 93 (14/15) 90 (9/10) 80 (8/10) 70 (7/10)
70 (7/105) 93 (14/10) 100 (10/1) 80 (8/10) 70 (7/10)
2.0 2.0 2.0 2.0 2.0 2.0
10 (2/20) 25 (5/20) 45 (9/20) 53 (8/15) 5 (1/20) 13 (2/16)
0 (0/20) 5 (1/20) 15 (3/20) 33 (5/15) 0 (0/20) 0 (0/16)
10 (2/20) 25 (5/20) 45 (9/20) 67 (10/15) 5 (1/20) 0 (0/16)
10 (2/20) 25 (5/20) 45 (9/20) 67 (10/15) 5 (I/20) 0 (0/16)
Reduced forms
L-Ascorbic acid L-Cysteine (FB) DL-Homocysteine (FB) L-Glutathione (Rd.) N-Acetyl-L-cysteine Oxidized forms
L-Dehydroascorbic acid L-Cystine DL-Homocystine L-Glutathione (Ox.) DL-Thioctic acid Other sulfur compounds
Thiamin- HC1 L-MTCA Taurine Cysteamine 9 HC1 Sodium metabisulfite L-Cysteic acid 9HzO
Compounds tested were intubated orally 30-45 minutes prior to oral intubation of 18 m M / k g of acetaldehyde (LD 90 dose). Doses are expressed in mM/kg. After intubation, rats were observed over a 24-72 hour period for anesthetic effects (loss of elevation and righting reflexes) and lethal response (deaths). Figures in parentheses = No. of rats anesthetized or dead/No, of rats tested. Anesthesia (loss of righting reflexes) with acetaldehyde generally occurred within 3-10 minutes and lasted for 5-15 minutes. Deaths were recorded at 3, 24, and 72 hours.
166
Protective Action of Ascorbic Acid and Sulfur Compounds
Table 2 Protective action of combinations of L-ascorbic acid and certain sulfur compounds against anesthetic and lethal effects of acetaldehyde in the rat. (Toxic oral dose of acetaldehyde used was LD 90 = 18 mM/kg.) Combinations (CB) tested (oral dose)
Dose (mM/kg)
CB1 : L-Cysteine FB Thiamin 9HC1 CB2: L-Cysteine FB Thiamin 9HCI CB3 : L-Ascorbic acid L-Cysteine FB Thiamin 9HCI CB4: L-Ascorbic acid N-Acetyl-L-cysteine Thiamin 9HC1
1.0 0.3 2.0 0.3 2.0 1.0 0.3 2.0 1.0 0.3
~ anesthetized after 3-10 minutes
~ dead after 3 hours
24 hours
72 hours
60 (6/10)
10 (1/10)
60 (6/10)
60 (6/10)
13 (2/15)
0 (0/15)
13 (2/15)
13 (2/15)
10 (3/30)
0 (0/30)
0 (0/30)
0 (0/30)
15 (3/20)
5 (1/20)
5 (1/20)
5 (1/20)
33 (5/15)
13 (2/15)
20 (3/15)
20 (3/15)
lntraperitoneal (i. p.) dose CB3 (i.p.): Composition same as CB3
Combinations of test compounds were prepared as single solutions in saline. Procedural details of intubation and observations made are the same as described in Table 1 footnote, except for Combination CB3 (i.p.). Combination CB3 (i.p.) was administered intraperitoneally once a day for four days prior to dosing with acetaldehyde. On the fourth day, acetaldehyde (18 mM/kg) was intubated orally 30--45 minutes after the last intraperitoneal injection of Combination CB3 (i.p.).
acetaldehyde at the LD 90 dose level carefully standarized for rats of the above strain, sex, age, weight, and overnight treatment and found to be 18 mM/kg as previously reported [1]. Doses were expressed in mM/kg. Rats were observed after intubation over a 72-hour period for anesthetic effects (loss of righting reflexes) and lethal response (deaths). Further details of the test procedure and data obtained are presented in Tables 1 and 2. The effect of using different routes of drug administration on protectant activity against acetaldehyde was also tested with a combination of ascorbic acid, cysteine, and thiamin (i.e., combination CB3 in Table 2). In this case, the protectant combination CB3 was administered intraperitoneally and the acetaldehyde orally. For details of procedure, see Table 2. All test compounds listed in Tables 1 and 2 were obtained from the NBC Division of ICN Pharmaceuticals, Inc., with the exceptions listed as follows: DLthioctic acid (Aldrich), taurine (Schwarz-Mann), cysteamine - HC1 (Eastman), sodium metabisulfite (Fisher), and L-MTCA (see Reference 1 for its preparation). Compounds not listed in Table 1 were from the following sources: BAL (Aldrich), DTT (NBC), and DTE (NBC). Acetaldehyde was obtained from the Eastman Kodak Company.
Results Results are self-evident from Table 1 a n d Table 2. F r o m Table 1, it is obvious that the reduced forms of L-ascorbic acid, L-cysteine FB, L-glutathione, a n d N-acetyl-L-cysteine gave m a r k e d protection in rats against the anesthetic a n d
lethal effects of acetaldehyde. Thus, 72 hours after treatment, lethal values were only 27% for L-ascorbic acid, 20 % for L-cysteine FB, a n d 33% for reduced g l u t a t h i o n e c o m p a r e d to 90% for saline controls. Complete protection (zero % lethality) was obtained with N-acetyl-L-cysteine,butonly meager protection with DL-homocysteine (60% lethality). Protection against transitory anesthetic effects was best o b t a i n e d with L-cysteine a n d its N-acetyl derivative (20% a n d 10% anesthetized, respectively) in c o m p a r i s o n with the saline c o n t r o l (96% anesthetized). The oxidized forms of these c o m p o u n d s showed very little p r o t e c t a n t activity against either anesthetic or lethal effects of acetaldehyde. Other sulfur c o m p o u n d s which gave virtually complete p r o t e c t i o n against anesthetic a n d lethal effects were s o d i u m metabisulrite (Na2S205) which resulted in only 5% lethality a n d L-cysteic acid in zero % lethality. O n the other hand, the amines t a u r i n e a n d cysteamine 9 HC1 showed m u c h less protection t h a n their c o r r e s p o n d i n g acids (L-cysteic acid a n d L-cysteine, respectively). P r e l i m i n a r y screening tests with c o m p o u n d s n o t listed in Table 1 gave the following results : B A L (2, 3 - d i m e r c a p t o - l - p r o panol) was f o u n d to be non-protective at 0.5 m M / kg, a dose n o t too far r e m o v e d from its lethal dose level of 1.0 m M / k g . O n the other h a n d , D T T (diothiothreitol) a n d D T E (diothioerythreitol) at
Protective Action of Ascorbic Acid and Sulfur Compounds a dose level of 0.5 mM/kg gave 50% protection with the lethal dose for both compounds found to be 2 mM/kg. Table 2 presents data on protection against acetaldehyde toxicity by certain combinations of above test compounds with ascorbic acid some of which at the doses used are more effective than any single component alone. At the dose levels tested, combinations of L-cysteine FB and thiamin- HC1 (CB1 and CB2) were not completely effective. The combination (CB3) of L-ascorbic acid (2 mM/kg), L-cysteine FB (1.0 mM/kg), and thiamin 9HC1 (0.3 mM/kg) was the most effective, giving complete protection (zero % lethality) for 72 hours in the 30 rats tested. Replacement of L-cysteine FB by N-acetyl-L-cysteine at an equivalent mole-dose level as in combination (CB4) gave virtually the same degree of protection (only 5% lethality). Note that in Table 2 data of CB1, CB2, CB3, and CB4 are results obtained only with oral intubation of the protectant-combinations and acetaldehyde. When the protectant-combination CB3 was administered for 4 days intraperitoneally (i.p.) and then challenged with the oral LD 90 dose of acetaldehyde, a high degree of protection against anesthetic and lethal effects was also observed. Protection obtained by i.p. administration of protectants, however, was somewhat less effective than protection obtained by their oral administration, resulting in 20% lethality vs. zero % lethality, respectively.
Discussion As pointed out previously [1], special precautions were taken to use an oral LD 90 dose (18 mM/kg) of freshly-distilled acetaldehyde with test rats carefully standardized with respect to strain, sex, age, weight, and overnight fast. An LD 90 dose and its careful standardization was necessary to minimize variability in results, particularly since acetaldehyde is so rapidly metabolized in the body. From this study, it is obvious that at high oral doses (2 mM/kg), L-ascorbic acid and certain sulfhydryl compounds give marked protection against acetaldehyde toxicity and lethality, whereas their oxidized counterparts do not. Notable for high protection in this group are L-cysteine and N-acetyl-L-cysteine and to a lesser extent reduced glutathione. Other sulfur compounds showing a high degree of protective activity at high doses are thiamin, L-2-methylthiazolidine-4-carboxylic acid (L-MTCA), L-cys-
167
teic acid, and sodium metabisulfite. Taurine showed protective activity to a lesser extent and cysteamine 9HC1 very little protection. The most active protectants at 2 mM/kg were: N-acetyl-Lcysteine, thiamin. HC1, sodium metabisulfite, and L-cysteic acid. Since L-ascorbic acid, L-cysteine, and thiamin are all naturally-occurring metabolites, certain combinations of these compounds were tested at reduced dosage for greater protective activity than that obtainable with the individual compounds alone. A combination of L-ascorbic acid (2 mM/kg), I.-cysteine (1 mM/kg), and thiamin (0.3 mM/kg) gave virtually complete protection as previously reported [1]. Replacement of L-cysteine by N-acetyl-L-cysteine in this combination gave practically the same protective effect. With this combination, different routes of drug administration (i.e., protectants given intraperitoneally followed by acetaldehyde given orally) also demonstrated the protective effect, but to a somewhat lesser degree than the oral route for both. To the best of our knowledge, our findings demonstrate for the first time that direct protective action against acetaldehyde toxicity and lethality can be obtained with certain naturally-occurring metabolites, namely L-ascorbic acid, L-cysteine, and thiamin, preferably in combination at reduced dose levels. Thus, our findings point the way to a possible buildup of natural protection against the chronic body insult of acetaldehyde arising from heavy drinking of alcohol and heavy smoking of cigarettes. We hasten to add, however, that such an implication from our findings must be further evaluated in animals before any extrapolation of it for human use can be considered. The last two sentences in the above paragraph require further elaboration. This can, perhaps, best be done by a survey of published reports already in the literature. These reports when pieced together support the rationale that ascorbic acid, cysteine, thiamin, and certain other sulfur compounds may be useful as long-term protectants against the chronic toxicity of acetaldehyde, which could develop with high alcohol intake and heavy cigarette smoking [4]. A brief review of these published reports is now presented in the following paragraphs. (a) L-Ascorbic acid as a possible protectant against acetaldehyde There is much literature evidence which warrants consideration of L-ascorbic acid as a possible protectant. L-ascorbic acid is known to
168 overcome the adverse symptoms [28] and the cardiac effects [29] of the disulfiram-ethanol reaction, which is presumably due to acetaldehyde [20]. Currently, the Physicians' Desk Reference of 1974 [30] recommends a high dose of ascorbic acid (1 g) for management of the severe effects of the disulfiram-ethanol reaction. Moreover, body levels of ascorbic acid are known to be depressed in alcoholics [31-34] and in heavy smokers on adequate dietary intake [35, 36]. Clinically, ascorbic acid levels chronically depressed over long time periods have been linked to cardiac disorders [37, 38], to atherosclerosis, particularly in association with smoking [39], and to deep-vein thrombosis [40], although these claims require further confirmation. Physiologically, such depressed levels could be induced by elevated acetaldehyde levels releasing catecholamines [5] which, in turn, stimulate increased utilization of ascorbic acid for their resynthesis [41]. Depressed ascorbic acid levels could also result from cigarette smoke per se which can oxidize ascorbic acid by way of a free radical intermediate [42]. Acetaldehyde in common with other aldehydes, might be involved in such an oxidation possibly by way of hemiacetal formation with the hydroxyl groups of ascorbic acid in the 2, 3 position. (b) L-cysteine as a possible protectant against acetaldehyde L-cysteine was also considered as a possible protectant for a number of reasons. As pointed out previously [1], cysteine by virtue of its (-SH) group can protect rats against the lethal pulmonary edema induced by thiourea compounds. It could act similarly to protect against the lethal pulmonary edema induced by acetaldehyde [43]. Additionally, cysteine and reduced glutathione by virtue of their (-SH) group are known to protect the phagocytoxic activity of alveolar macrophages from inhibition by cigarette smoke [44], presumably by protecting glyceraldehyde-3phosphate dehydrogenase activity [45]. This enzyme like liver aldehyde dehydrogenase is a nonspecific thiol-requiring enzyme capable of oxidizing acetaldehyde [46]. These observations further confirm the idea that cigarette smoke has oxidant activity [44] which can be prevented by sulfhydryl compounds [45] and possibly also by ascorbic acid [42]. Chemically, L-cysteine by way of its (-SH) group complexes with acetaldehyde to form L-2-methylthiazolidine-4-carboxylic acid (L-MTCA) by way of an intermediary hemiacetal
Protective Action of Ascorbic Acid and Sulfur Compounds or Schiff base, the latter being the favored intermediate [47]. As previously suggested [1], L-MTCA might then serve as a non-toxic metabolic detoxification product as well as a protectant by generating free intracellular (-SH) groups to complex with acetaldehyde more effectively [48]. Biochemically, it has already been suggested that cysteine can protect Coenzyme A against acetaldehyde inhibition by complexing preferentially with acetaldehyde [49]. Presumably, reduced glutathione, homocysteine, and N-acetyl-cysteine could also complex with acetaldehyde by virtue of their free (-SH) groups [47]. N-acetyl-L-cysteine is a well-known mucolytic agent capable of liquefying mucus and secretions associated with pulmonary disorders [50]. It is used in human therapy and is currently listed in Physicians' Desk Reference of 1974 [51]. Finally, cysteine may be important in normal cardiac function by giving rise to taurine which has been claimed to be a regulator of cell potassium in the heart [52, 53]. (c) Thiamin as a possible protectant against acetaldehyde Thiamin could also serve as a protectant against acetaldehyde for some very good reasons. Thus, as cocarboxylase (pyrophosphate) it is believed to complex normally with acetaldehyde to eventually form acetyl Coenzyme A by way of the lipoic (thioctic) acid pathway [54]. Impairment of thiamin absorption known to occur in alcoholism [55] could thus predispose to elevated acetaldehyde levels and possibly lead thereby eventually to cardiomyopathy [56]. In this connection, thiamin has long been known to be a specific therapeutic in alcoholic beriberi heart disease [56], but its value in alcoholic cardiomyopathy is less certain [56]. (d) Possible metabolic interrelationships between L-ascorbic acid, L-cysteine, and thiamin From the literature, it is also evident that certain combinations of ascorbic acid, cysteine, and thiamin could function more effectively than the individual compounds alone as protectants against the chronic toxicity of acetaldehyde. HUEPER [57], as early as 1943 in studies relating to smoking, found that dietary supplements of ascorbic acid and cystine gave rats definite protection against chronic nicotine poisoning. He then concluded that 'exogenous and possibly dietary factors may account in part for the differences in individual susceptibility to nicotine in
Protective Action of Ascorbic Acid and Sulfur Compounds man and in animals'. Metabolically, ascorbic acid, cysteine, and thiamin are known to be interrelated in ways which could enhance their individual activity in the body. For example, thiamin is known to form with cysteine a mixed disulfide which has been claimed to be a more active form of thiamin in vivo [58]. Formation of this disulfide could be effected by the cysteine-cystine redox equilibrium system which in turn is believed to be regulated by the ascorbic-dehydroascorbic acid redox system like that of glutathione [59]. This possibility is supported by reports that ascorbic acid has a thiamin-sparing action in vivo which may be due to thiamin disulfide activation in tissues by cysteine [60, 61]. Thus, the ascorbic-dehydroascorbic acid redox system may enhance thiamin activity by activating the redox systems of cysteine or glutathione to form with thiamin the more active thiamin-cysteine disulfide. Despite an element of controversy [62], the practical import of these observations is apparent from other reports that the disulfides of thiamin, notably allithiamine [63] and especially thiamin propyl disulfide [64], are more active, better absorbed, and better utilized than thiamin itself. Indeed, in alcoholic patients, thiamin propyl disulfide may correct thiamin depletion syndromes which are resistant to oral thiamine hydrochloride [65]. Finally, it is important, too, to remember that cysteine by complexing with acetaldehyde may protect the (-SH) groups of Coenzyme A and reduced lipoic acid, both of which are associated with thiamin pyrophosphate activity. Thus, combinations of ascorbic acid, cysteine, and thiamin in certain ratios could function more effectively than any of these compounds alone as protectants against acetaldehyde.
(e) Sodium metabisulfite, L-cysteic acid, and taurine as protectants against acetaldehyde From the literature, it is evident that other sulfur compounds, particularly sodium metabisulfite, L-cysteic acid, and taurine could be effective as protectants against acetaldehyde. Thus, sodium metabisulfite (Na~S20~) was recently found to protect animals (cats, mice) from acetaldehyde poisoning and alcoholic patients from the disulfiram-ethanol reaction [6@ Our findings with rats not only confirm the highly protective action of sodium metabisulfite against acetaldehyde but also reveal cysteic acid to be equally effective. Presumably, protection results from acetaldehyde complexing with the sulfite formed
169 from sodium metabisulfite or with cysteic acid. Taurine, which should presumably be equally as active, however, showed only moderate protective activity. This finding might be explained on the basis of the following speculation which also directs attention to the possible importance of taurine in alcoholic cardiomyopathy. Thus, taurine has been reported: (a) to be markedly increased in the urine of patients with acute alcoholism [67] and (b) to prevent and even reverse ventricular arrhythmias in the heart induced by epinephrine (catecholamine) by way of loss of myocardial potassium [52, 53]. Since disturbances of this nature (ventricular arrhythmias and loss of potassium) also occur in alcoholic cardiomyopathy [68], taurine could conceivably be of value in this condition. However, uptake of taufine into rat heart is slow, reaching a maximum in 3 or more days [69]. Therefore, its metabolic precursors, cysteine and cysteic acid [52], might be more effective in maintaining necessary levels of it for proper cardiac functioning. This speculation could explain the greater effectivity of cysteine and cysteic acid than taurine in our protection experiments reported herein. More experimental work is necessary, however, before any definite conclusions can be drawn.
(f) Acetaldehyde formation and binding by tissues in relation to its chronic toxicity and the implications thereof Some final discussion concerning the chronic toxicity effects of acetaldehyde is in order. Only recently have the adverse tissue effects of prolonged exposure to acetaldehyde begun to be perceived in relation to certain disease states in the cardiovascular, respiratory, and central nervous systems as well as in the aging process. Perhaps this is so because blood acetaldehyde levels when increased to measurable amounts with heavy alcohol intake remain relatively low compared with blood ethanol concentrations and show no dose nor dose-time relationships to blood ethanol concentrations [6]. The presumed explanation is that acetaldehyde is rapidly metabolized chiefly in the liver and that only a small excess passes into the blood. Blood levels thus merely reflect the total body concentration of excess free acetaldehyde not destroyed in the liver. However, three important considerations are thereby overlooked: (a) acetaldehyde formation from ethanol in tissues other than liver [70, 71], (b) the existence of acetaldehyde in blood and other tissues in two forms:
170 free and chemically-bound [72], and (c) acetaldehyde inhalation from cigarette smoke about which very little is presently known [9]. Concerning acetaldehyde formation in tissues other than liver, the enzymes catalyzing ethanol metabolism (alcohol dehydrogenase and aldehyde dehydrogenase) have been shown to be widely distributed in mammalian tissues [70, 71 ]. In the rat, these enzymes are found in a number of neural and somatic tissues known to be vulnerable to excessive ethanol in man [70]. Although the level of these enzymes in liver is highest by far, they do occur in other tissues at levels probably sufficient for local tissue metabolism of ethanol [70]. It is probable, therefore, that ethanol can be transported by the blood to tissues other than liver and metabolized therein to form the more toxic acetaldehyde. The vulnerability to acetaldehyde, as well as to ethanol, of all body tissues including liver deserves further study. Concerning the chemical-binding of acetaldehyde in blood and other tissues, evidence is rapidly accumulating of the importance of such occurrence. In blood, such binding was recently rediscovered in human erythrocytes and found to be released by precipitation or hemolysis in the presence of ethanol [73]. Serum binding of acetaldehyde was believed to be involved in the rapid incorporation of ethanol-l-14C into serum proteins [74]. Presumably, the ethanol forms acetaldehyde which then reacts with certain functional groups in proteins, e.g. free amino or free sulfhydryl groups [74]. In tissues, acetaldehyde can bind with liver and brain Coenzyme A with resultant loss in activity of the latter [75]. Acetaldehyde binding may also take part in the inhibition of certain mitochondrial functions, such as energized calcium uptake [76]. Very recently, acetaldehyde was found to inhibit cardiac microsomal protein synthesis in the guinea-pig at concentrations found in man after moderate ethanol ingestion [12]. On this basis it was speculated that such decreased protein synthesis could play a role in alcoholic cardiomyopathy [12]. F r o m the foregoing, it is obvious that evidence is increasing for the binding of acetaldehyde in blood and other tissues with possibly far reaching consequences. The potential of acetaldehyde for binding chemically with tissues has far reaching implications with respect to its chronic toxicity. Theoretically, acetaldehyde can combine with various functional groups found in proteins (amino, sulf-
Protective Action of Ascorbic Acid and Sulfur Compounds
hydryl, hydroxyl groups, etc.). Biochemically, it has been demonstrated to give a 'browning reaction' with proteins, involving amino groups, guanidyl groups, arid crosslinkage formation [77]. Immunologically, it like formaldehyde, has been found to act at low concentrations on antibodies [78, 79] and presumably also on antigens [80]. With certain antibodies (antipneumococcus serum and diphtheria antitoxin), solubilization of the antibody by the aldehydic group occurs with resultant loss of antibody protective power in the living animal [78, 79]. With certain antigens (diphtheria toxin), the aldehydic group produces toxoids by inactivating toxic groups [80]. It is important to note that this latter reaction is a slow and irreversible one [81]. It is not unreasonable therefore to suppose that acetaldehyde (and other aldehydes) can react with proteins slowly and irreversibly to result in adverse functional changes in these proteins, for example in reduced enzymatic activity or reduced metal-binding capacity. Such changes slow and irreversible at the molecular IeveI over a period of years could lead to adverse tissue changes and eventually organ dysfunction. In this context, our findings suggest that certain body compounds capable of protecting against acetaldehyde toxicity could lead to a possible build-up of natural protection against the chronic body insult of acetaldehyde arising from heavy alcohol intake and cigarette smoking. Such protection might be developed by increasing the levels of certain body metabolites reacting directly with, or involved in the enzymatic destruction of, acetaldehyde and other aldehydes. Candidates for such long-term protection are •-ascorbic acid possibly in combination with certain sulfur compounds, notably L-cysteine, L-cysteic acid, and thiamin. Further research in animals is necessary, however, before the long-term use of these compounds in high doses for human beings can be considered. Received 22 February 1975.
References [1] H. SPRINCE, C. M. PARKER,G. G. SMITHand L. J. GONZALES, Protection against Acetaldehyde Toxicity in the
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Protective Action of Ascorbic Acid and Sulfur Compounds
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172 [40] C.R. SPITTLE, Vitamin C and Deep- Vein Thrombosis, Lancet 2, 199-201 (1973). [41 ] S. KAUFMAN, Coenzymes andHydroxylases: Ascorbate and Dopamine-fl-Hydroxylase; Tetrahydropteridines and Phenylalanine and Tyrosine HydroxyIases, Pharmac. Rev. 18, 61-69 (1966). [42] M.H. BILIMORIA and M. A. NISBET, Effect of Tobacco Smoke Condensates on Ascorbate, Beitr. Tabakforsch. 6 (1), 32-35 (July 1971). [43] J. AKABANE, Aldehydes and Related Compounds, in: International Encyclopedia of Pharmacology and Therapeutics, vol. II (Ed. J. Tr6moli6res; Pergamon Press, New York 1970), p. 523-560. [44] G. M. GREEN, Cigarette Smoke: Protection of Alveolar Macrophages by Glutathione and Cysteine, Science 162, 810-811 (1968). [45] G. M. POWELL and G . M . GREEN, Cigarette Smoke A Proposed Metabolic Lesion in Alveolar Macrophages, Biochem. Pharmac. 21, 1785-1798 (1972). [46] H. WEINER, P. KING, J. H.J. HI: and W.R. BENSCH, Mechanistic and Enzymatic Properties of Liver Aldehyde Dehydrogenases, in: Alcohol and Aldehyde Metabolizing Enzymes (Eds R. G. Thurman, T. Yonetani, J. R. Williamson and B. Chance; Academic Press, Inc. 1974), p. 101-113. [47] M. FRIEDMAN, The Chemistry and Biochemistry of the Sulfhydryl Group in Amino Acids, Peptides, and Proteins (Pergamon Press, New York 1973), p. 88-90, 92-95. [48] H.J. DEBEY, J.B. MACKENZIE and C.G. MACKENZIE, The Replacement by Thiazolidinecarboxylic AcM of Exogenous Cystine and Cysteine, J. Nutr. 66, 607-619 (1958). [49] G.J. MARTIN, J.N. MOSS, R.D. SMYTH and H. BECK, The Effect of Cysteine in Modifying the Action of Ethanol Given Chronically in Rats, Life Sci. 5, 2357-2362 (1966). [50] M. FRIEDMAN, The Chemistry and Biochemistry of the Sulfhydryl Group in Amino Acids, Peptides, and Proteins (Pergamon Press, New York 1973), p. 418. [51] Physicians' Desk Reference, 28th ed. (Pub. C.E. Baker, Jr. ; Med. Economics Co., Oradell, New Jersey 1974), p. 981. [52] J.G. JACOBSENand L. H. SMITH, Jr., Biochemistry and Physiology of Taurine and Taurine Derivatives, Physiol. Rev. 48, 424-511 (1968). [53] W.O. READ and J.D. WELTY, Taurine as a Regulator of Cell Potassium in the Heart, in: Electrolytes and Cardiovascular Diseases, vol. 1 (Ed. E. Bajusz; S. Karger Basel-New York 1965), p. 70-85. [54] R.E. OLSON, Nutrition and Alcoholism, in: Modern Nutrition in Health and Disease (Eds M.G. Wohl and R.S. Goodhart; Lea and Febiger 1968), p. 767-781. [55] P. A. TOMASULO, R. M. H. KATER and F. L. IBER, Impairment of Thiamin Absorption in Alcoholism, Am. J. Clin. Nutr. 21, 1341-1344 (1968). [56] G. E.BURCH and T. D.GILES, Alcoholic Cardiomyopathy, in: The Biology of Alcoholism, vol. 3, chapter 13 (Eds B. Kissin and H. Begleiter; Plenum Press, New York 1974), p. 435-460.
Protective Action of Ascorbic Acid and Sulfur Compounds
[57] W~ C. HUEPER, Experimental Stadies in Cardiovascular Pathology. VIII. Chronic Nicotine Poisoning in Rats and in Dogs, Arch. Path. 35, 846-856 (1943). [58] T. MATSUKAWAand S. YURUOI, On a New Derivative of Thiamine with Cysteine, Science 118, 109-111 (1953). [59] B. VENNESLAND and E. E. CONN, The Enzymatic Oxidation and Reduction of Glutathione, in: Glutathione, A Symposium (Eds S. Colowick et al. ; Academic Press, New York 1954), p. 105-127. [60] Z.G. B~,NI-IIDI, Thiamine-Sparing Action of Ascorbie Aeid on some Lactobaeilli and the Rat Due to Thiamine Disulfide Activation, Ann. N.Y. Acad. Sci. 92, 324-332 (1961). ]61] Z.G. BXNI-1IDI, Molecular Physiology of Thiamine, Acta physiol, scand. 50, Suppl. 174 (20 p.) (1960). [62] D.S. MURDOCK, M. L. DONALDSON and C. J. GUBLER, Studies on the Mechanism of the 'Thiamin-Sparing' Effect of Aseorbic Acid in Rats, Am. J. Clin. Nutr. 27, 696-699 (1974). [63] M. FUJIWARA, H. NANJO, T. ARAI and J. SuzooKI, Allithiamine, a Derivative of Vitamin B1.11. Effect of Allithiamine on the Living Organism~ J. Biochem. (Japan) 41, 273-285 (1954). [64] D. HIOCO, R. TIXIER and A. UZAN, Biochemical Investigation of Thiamin Propyl Disulfide. Its Transformation in vivo into Cocarboxylase, Bull. Soc. Chim. Biol. 41, 1075-1083 (1959). [65] A. D. THOMSON, O. FRANK, H. BAKER and C. M. LEEVY, Thiamin Propyl Disulfide: Absorption and Utilization, Ann. Int. Med. 74, 529-534 (1971). [66] G. I. YEZRIYEEEV, Atsetal'degid i alkogolizm; farmakogenez Teturamalkogol'noi reaktsii i upravleniye eyu putem svyazyvaniya atsetal'degida metabisul'fitom natriya (Acetaldehyde and Alcoholism; Pharmacogenesis of the Teturam-Alcohol Reaction and Its Control through Compound Formation between Acetaldehyde and Sodium Metabisulfite), Zh.Nevropat. Psikhiat. 73, 238-243 (1973). [67] F.P. TURNER and V. C. BRUM, The Urinary Excretion of Free Taurine in Acute and Chronic Disease, Following Surgical Trauma, and in Patients with Acute Alcoholism, J. Surg. Res. 4, 423-431 (1964). [68] T.J. REGAN, Ethyl Alcohol and the Heart, Circulation 44, 957-963 (1971). [69] D. G. SPAETH and D. L. SCHNEIDER, Turnover of Taurine in Rat Tissues, J. Nutr. 104, 179-186 (1974). [70] N.H. RASKIN and L. SOKOLOFF, Enzymes Catalysing Ethanol Metabolism in Neural and Somatic Tissues of the Rat, J. Neurochem. 19, 273-282 (1972). [71] R. A. DEITRICI-I, Tissue and Subcellular Distribution of Mammalian Aldehyde-Oxidizing Capacity, Biochem. Pharmac. 15, 1911-1922 (1966). [72] H. P. T. AMMON,Discussion on Pages 226-227 of Paper by E. G. Truitt, Jr., Entitled Blood Acetaldehyde Levels after Alcohol Consumption by Alcoholic and Nonalcoholic Subjects, in: Biological Aspects of Alcohol, chapter 9 (Eds M. K. Roach, W. M. Mclsaac and P.J. Creaven; University of Texas Press, Austin 1971 ), p. 212-232.
Protective Action of Ascorbic Acid and Sulfur Compounds
[73] E.B. TRUITT,Ethanol-lnduced Release of Acetaldehyde from Blood and lts Effect on the Determination of Acetaldehyde, Q. J. Stud. Alcohol 31, 1-12 (1970). [74] S. GUDBJARNASONand A.S. PRASAO, Cardiac Metabolism in Experimental Alcoholism, in: Biochemical and Clinical Aspects of Alcohol Metabolism, chapter 26 (Ed. V.M. Sardesai; Charles C. Thomas, Springfield 1969), p. 266-272. [751 H.P.T. AMMON,C.-J. ESTLERand F. HELM,Effect of Alcohol and Acetaldehyde on Coenzyme A, in: Biological Aspects of Alcohol, chapter 8 (Eds M.K. Roach, W.M. McIsaac and P.J. Creaven; University of Texas Press, Austin 1971), p. 185-211. [76] E. RUBIN and A.I. CEDERBAUM,Effects of Chronic Ethanol Feeding and Acetaldehyde on Mitochondrial Functions and the Transfer of Reducing Equivalents, in: Alcohol and Aldehyde Metabolizing Systems (Eds R.G. Thurman, T. Yonetani, J.R. Williamson and B. Chance; Academic Press, New York 1974), p. 435-455.
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Agents and Actions vol. 5/2 (1975) Birkh/iuser Verlag, Basel
Protective Action of Ascorbic Acid and Sulfur Compounds against Acetaldehyde Toxicity: Implications in Alcoholism and Smoking 1) by HERBERT SPRINCE2), CLARENCE M. PARKER, GEORGE G. SMITH and LEON J. GONZALES U.S. Veterans Administration Hospital, Coatesville, Pennsylvania 19320, and The Departments of Psychiatry and Human Behavior and Pharmacology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA
Abstract Acetaldehyde is a toxic substance common to heavy drinking of alcohol and heavy smoking of cigarettes. It has been implicated thereby in diseases of the cardiovascular, respiratory, and central nervous systems. Protection against acetaldehyde toxicity (i.e. anesthesia and lethality) was studied in rats by oral intubation of test compounds 30-45 minutes prior to oral intubation of a standardized oral LD 90 dose (18 millimoles/kilogram) of acetaldehyde. Animals were monitored for anesthesia (loss of righting reflexes) and lethality for 72 hours. A total of 18 compounds was tested. L-ascorbic acid at 2 miRimoles/kilogram (mM/kg) showed moderate protection against anesthesia and marked protection against lethality. Greatest protection against anesthesia and lethality was obtained at 2 mM/kg with each of the following: L-eysteine, N-acetyI-L-cysteine, thiamin" HCI, sodium metabisulfite, and L-eysteie acid. A combination of L-aseorbic acid with L-cysteine, and thiamin.HCl at reduced dose levels (2.0, 1.0 and 0.3 mM/kg, respectively) gave virtually complete protection. A detailed literature review is presented of the rationale and significance of these findings. Our findings could point the way to a possible build-up of natural protection against the chronic body insult of acetaldehyde arising from heavy drinking of alcohol and heavy smoking of cigarettes.
Preceding publications from this laboratory [1-3] directed attention to a possible means of protection against the adverse effects of acetaldehyde, a key intermediary metabolite of ethyl alcohol (ethanol), more toxic than ethanol, and also a toxic component of cigarette smoke. Currently, the chronic adverse effects of acetaldehyde on body tissues assume even greater significance with the increasing realization that, ~) Supported by U.S. Veterans Administration (Project No. 8078-01 and 8078-04). ,z) Address reprint requests to Dr. Herbert Sprince, VA Hospital, Coatesville, Pennsylvania 19320, USA.
despite its rapid metabolism in vivo, body levels of acetaldehyde may be elevated by both heavy alcohol drinking [4-6] and heavy cigarette smoking [4, 7-9]. This possible relationship of acetaldehyde to the adverse effects of both alcohol and cigarette smoking was clearly stated in the 1970 review of T. N. JAMES et al. [4], and is strengthened by recent reports establishing that heavy drinkers are generally also heavy smokers [10, 11 ]. In the case of alcoholism, acetaldehyde has been implicated in alcoholic cardiomyopathy [4, 12-14], in alcohol addiction [15, 16], in ethnic sensitivity to alcohol [17], in alcoholic cerebellar degeneration [18], in the aging process [19], and in the disulfiram-ethanol reaction [20] used in the aversion treatment of alcoholism. Medically, this last named reaction is of greater importance than generally realized since a disulfiram-like interaction with ethanol also occurs with a number of therapeutic drugs other than disulfiram [21]. Acetaldehyde could also play a role in the development of 'alcoholic lung disease' [22-24]. In the case of heaw cigarette smoking, acetaldehyde has been implicated not only in cardiac disease [12-14] but also in pulmonary disease by way of its highly toxic action on cilia of the respiratory tract [25], an effect believed to be related to the premalignant state of the tracheobronchial tree [26, 27]. More recently, its content in cigarette smoke in association with cigarette tar has been found to correlate highly with the 'free lung cell response' of RYLANDER [8]. This response is defined as the change in numbers of macrophages and leucocytes induced by cigarette smoke and 'probably represents a significant exposure reaction in terms of development of pulmonary disease' (quotation from RYLANDER [8]). From
Protective Action of Ascorbic Acid and Sulfur Compounds
the f o r e g o i n g , it is e v i d e n t t h a t a c e t a l d e h y d e is a toxic s u b s t a n c e c o m m o n to h e a v y a l c o h o l d r i n k i n g a n d h e a v y cigarette s m o k i n g a n d i m p l i c a t e d i n c a r d i o v a s c u l a r a n d p u l m o n a r y disease [4]. T h e r e f o r e it b e c o m e s i m p o r t a n t to s e a r c h for i n vivo p r o t e c t a n t s a g a i n s t its a d v e r s e effects. S u c h protectants (particularly, naturally-occurring ones) c o u l d be o f p o t e n t i a l t h e r a p e u t i c v a l u e as p r e v e n t i v e s for tissues c h r o n i c a l l y e x p o s e d to elevated a c e t a l d e h y d e levels. I n t h e p r e v i o u s p u b l i c a t i o n s f r o m this l a b o r a t o r y [1-3], details were p r e s e n t e d o f t h e protective a c t i o n i n rats o f L-cysteine free base (FB), t h i a m i n - H C I , a n d L - 2 - m e t h y l t h i a z o l i d i n e 4 - c a r b o x y l i c acid a g a i n s t a c e t a l d e h y d e toxicity a n d lethality at a carefully s t a n d a r d i z e d L D 90 dose level. T h i s s t u d y p r e s e n t s o u r f i n d i n g s o n the protective a c t i o n o f r e l a t e d s u l f u r c o m p o u n d s a n d a s c o r b i c acid a g a i n s t a c e t a l d e h y d e toxicity in rats at the s a m e s t a n d a r d i z e d L D 90 dose level.
165
Materials and methods
The following is a brief outline of the methodology used; details are set forth in our previous publication [1]. Carworth Farms (CFE) strain male albino rats 90 4- 5 days old and weighing 365 4- 20 grams were deprived of food, but not water, overnight. Next morning, they were reweighed just prior to test for calculation of dosages of the test compounds. Compounds tested and the doses used are listed in Tables 1 and 2 under 'results'. Test compounds were freshly prepared as solutions or fine suspensions in physiological (0.95/00)saline purged with nitrogen gas to protect against undesirable oxidation. Dilutions were made to concentrations convenient for oral intubation of the desired dose at convenient volumes (1-2.5 ml). Acetaldehyde was always freshly distilled just before use. For initial screening, 30 rats per day were generally tested in equal groups (5 rats/group). Such tests were made in duplicate on two consecutive days to give a minimum of 10 rats/group tested. When a given test compound showed definite protection, these tests were replicated with more rats to the total number indicated in Table 1. Test compounds or combinations thereof were intubated orally 30-45 minutes prior to oral intubation of
Table 1
Protective action of L-ascorbic acid and sulfur compounds against anesthetic and lethal effects of acetaldehyde in the rat. (Toxic oral dose of acetaldehyde used was LD 90 = 18 mM/kg.) Compounds tested (oral dose)
Dose (mM/kg)
~ anesthetized after 3-10 minutes
~ dead after 3 hours
24 hours
72 hours
Control (saline)
-
96 (48/50)
54 (27/50)
90 (45/50)
90 (45/50)
2.0 2.0 2.0 2.0 2.0
53 (8/15) 20 (5/25) 60 (6/10) 47 (7/15) 10 (2/20)
27 (4/15) 8 (2/25) 50 (5/10) 0 (0/15) 0 (0/20)
27 (4/15) 20 (5/25) 60 (6/10) 33 (5/15) 0 (0/20)
27 (4/15) 20 (5/25) 60 (6/10) 33 (5/15) 0 (0/20)
2.0 2.0 2.0 2.0 2.0
80 (8/10) 87 (13/15) 90 (9/10) 80 (8/10) 60 (6/10)
20 (2/10) 47 (7/15) 30 (3/10) 10 (1/10) 60 (6/10)
70 (7/10) 93 (14/15) 90 (9/10) 80 (8/10) 70 (7/10)
70 (7/105) 93 (14/10) 100 (10/1) 80 (8/10) 70 (7/10)
2.0 2.0 2.0 2.0 2.0 2.0
10 (2/20) 25 (5/20) 45 (9/20) 53 (8/15) 5 (1/20) 13 (2/16)
0 (0/20) 5 (1/20) 15 (3/20) 33 (5/15) 0 (0/20) 0 (0/16)
10 (2/20) 25 (5/20) 45 (9/20) 67 (10/15) 5 (1/20) 0 (0/16)
10 (2/20) 25 (5/20) 45 (9/20) 67 (10/15) 5 (I/20) 0 (0/16)
Reduced forms
L-Ascorbic acid L-Cysteine (FB) DL-Homocysteine (FB) L-Glutathione (Rd.) N-Acetyl-L-cysteine Oxidized forms
L-Dehydroascorbic acid L-Cystine DL-Homocystine L-Glutathione (Ox.) DL-Thioctic acid Other sulfur compounds
Thiamin- HC1 L-MTCA Taurine Cysteamine 9 HC1 Sodium metabisulfite L-Cysteic acid 9HzO
Compounds tested were intubated orally 30-45 minutes prior to oral intubation of 18 m M / k g of acetaldehyde (LD 90 dose). Doses are expressed in mM/kg. After intubation, rats were observed over a 24-72 hour period for anesthetic effects (loss of elevation and righting reflexes) and lethal response (deaths). Figures in parentheses = No. of rats anesthetized or dead/No, of rats tested. Anesthesia (loss of righting reflexes) with acetaldehyde generally occurred within 3-10 minutes and lasted for 5-15 minutes. Deaths were recorded at 3, 24, and 72 hours.
166
Protective Action of Ascorbic Acid and Sulfur Compounds
Table 2 Protective action of combinations of L-ascorbic acid and certain sulfur compounds against anesthetic and lethal effects of acetaldehyde in the rat. (Toxic oral dose of acetaldehyde used was LD 90 = 18 mM/kg.) Combinations (CB) tested (oral dose)
Dose (mM/kg)
CB1 : L-Cysteine FB Thiamin 9HC1 CB2: L-Cysteine FB Thiamin 9HCI CB3 : L-Ascorbic acid L-Cysteine FB Thiamin 9HCI CB4: L-Ascorbic acid N-Acetyl-L-cysteine Thiamin 9HC1
1.0 0.3 2.0 0.3 2.0 1.0 0.3 2.0 1.0 0.3
~ anesthetized after 3-10 minutes
~ dead after 3 hours
24 hours
72 hours
60 (6/10)
10 (1/10)
60 (6/10)
60 (6/10)
13 (2/15)
0 (0/15)
13 (2/15)
13 (2/15)
10 (3/30)
0 (0/30)
0 (0/30)
0 (0/30)
15 (3/20)
5 (1/20)
5 (1/20)
5 (1/20)
33 (5/15)
13 (2/15)
20 (3/15)
20 (3/15)
lntraperitoneal (i. p.) dose CB3 (i.p.): Composition same as CB3
Combinations of test compounds were prepared as single solutions in saline. Procedural details of intubation and observations made are the same as described in Table 1 footnote, except for Combination CB3 (i.p.). Combination CB3 (i.p.) was administered intraperitoneally once a day for four days prior to dosing with acetaldehyde. On the fourth day, acetaldehyde (18 mM/kg) was intubated orally 30--45 minutes after the last intraperitoneal injection of Combination CB3 (i.p.).
acetaldehyde at the LD 90 dose level carefully standarized for rats of the above strain, sex, age, weight, and overnight treatment and found to be 18 mM/kg as previously reported [1]. Doses were expressed in mM/kg. Rats were observed after intubation over a 72-hour period for anesthetic effects (loss of righting reflexes) and lethal response (deaths). Further details of the test procedure and data obtained are presented in Tables 1 and 2. The effect of using different routes of drug administration on protectant activity against acetaldehyde was also tested with a combination of ascorbic acid, cysteine, and thiamin (i.e., combination CB3 in Table 2). In this case, the protectant combination CB3 was administered intraperitoneally and the acetaldehyde orally. For details of procedure, see Table 2. All test compounds listed in Tables 1 and 2 were obtained from the NBC Division of ICN Pharmaceuticals, Inc., with the exceptions listed as follows: DLthioctic acid (Aldrich), taurine (Schwarz-Mann), cysteamine - HC1 (Eastman), sodium metabisulfite (Fisher), and L-MTCA (see Reference 1 for its preparation). Compounds not listed in Table 1 were from the following sources: BAL (Aldrich), DTT (NBC), and DTE (NBC). Acetaldehyde was obtained from the Eastman Kodak Company.
Results Results are self-evident from Table 1 a n d Table 2. F r o m Table 1, it is obvious that the reduced forms of L-ascorbic acid, L-cysteine FB, L-glutathione, a n d N-acetyl-L-cysteine gave m a r k e d protection in rats against the anesthetic a n d
lethal effects of acetaldehyde. Thus, 72 hours after treatment, lethal values were only 27% for L-ascorbic acid, 20 % for L-cysteine FB, a n d 33% for reduced g l u t a t h i o n e c o m p a r e d to 90% for saline controls. Complete protection (zero % lethality) was obtained with N-acetyl-L-cysteine,butonly meager protection with DL-homocysteine (60% lethality). Protection against transitory anesthetic effects was best o b t a i n e d with L-cysteine a n d its N-acetyl derivative (20% a n d 10% anesthetized, respectively) in c o m p a r i s o n with the saline c o n t r o l (96% anesthetized). The oxidized forms of these c o m p o u n d s showed very little p r o t e c t a n t activity against either anesthetic or lethal effects of acetaldehyde. Other sulfur c o m p o u n d s which gave virtually complete p r o t e c t i o n against anesthetic a n d lethal effects were s o d i u m metabisulrite (Na2S205) which resulted in only 5% lethality a n d L-cysteic acid in zero % lethality. O n the other hand, the amines t a u r i n e a n d cysteamine 9 HC1 showed m u c h less protection t h a n their c o r r e s p o n d i n g acids (L-cysteic acid a n d L-cysteine, respectively). P r e l i m i n a r y screening tests with c o m p o u n d s n o t listed in Table 1 gave the following results : B A L (2, 3 - d i m e r c a p t o - l - p r o panol) was f o u n d to be non-protective at 0.5 m M / kg, a dose n o t too far r e m o v e d from its lethal dose level of 1.0 m M / k g . O n the other h a n d , D T T (diothiothreitol) a n d D T E (diothioerythreitol) at
Protective Action of Ascorbic Acid and Sulfur Compounds a dose level of 0.5 mM/kg gave 50% protection with the lethal dose for both compounds found to be 2 mM/kg. Table 2 presents data on protection against acetaldehyde toxicity by certain combinations of above test compounds with ascorbic acid some of which at the doses used are more effective than any single component alone. At the dose levels tested, combinations of L-cysteine FB and thiamin- HC1 (CB1 and CB2) were not completely effective. The combination (CB3) of L-ascorbic acid (2 mM/kg), L-cysteine FB (1.0 mM/kg), and thiamin 9HC1 (0.3 mM/kg) was the most effective, giving complete protection (zero % lethality) for 72 hours in the 30 rats tested. Replacement of L-cysteine FB by N-acetyl-L-cysteine at an equivalent mole-dose level as in combination (CB4) gave virtually the same degree of protection (only 5% lethality). Note that in Table 2 data of CB1, CB2, CB3, and CB4 are results obtained only with oral intubation of the protectant-combinations and acetaldehyde. When the protectant-combination CB3 was administered for 4 days intraperitoneally (i.p.) and then challenged with the oral LD 90 dose of acetaldehyde, a high degree of protection against anesthetic and lethal effects was also observed. Protection obtained by i.p. administration of protectants, however, was somewhat less effective than protection obtained by their oral administration, resulting in 20% lethality vs. zero % lethality, respectively.
Discussion As pointed out previously [1], special precautions were taken to use an oral LD 90 dose (18 mM/kg) of freshly-distilled acetaldehyde with test rats carefully standardized with respect to strain, sex, age, weight, and overnight fast. An LD 90 dose and its careful standardization was necessary to minimize variability in results, particularly since acetaldehyde is so rapidly metabolized in the body. From this study, it is obvious that at high oral doses (2 mM/kg), L-ascorbic acid and certain sulfhydryl compounds give marked protection against acetaldehyde toxicity and lethality, whereas their oxidized counterparts do not. Notable for high protection in this group are L-cysteine and N-acetyl-L-cysteine and to a lesser extent reduced glutathione. Other sulfur compounds showing a high degree of protective activity at high doses are thiamin, L-2-methylthiazolidine-4-carboxylic acid (L-MTCA), L-cys-
167
teic acid, and sodium metabisulfite. Taurine showed protective activity to a lesser extent and cysteamine 9HC1 very little protection. The most active protectants at 2 mM/kg were: N-acetyl-Lcysteine, thiamin. HC1, sodium metabisulfite, and L-cysteic acid. Since L-ascorbic acid, L-cysteine, and thiamin are all naturally-occurring metabolites, certain combinations of these compounds were tested at reduced dosage for greater protective activity than that obtainable with the individual compounds alone. A combination of L-ascorbic acid (2 mM/kg), I.-cysteine (1 mM/kg), and thiamin (0.3 mM/kg) gave virtually complete protection as previously reported [1]. Replacement of L-cysteine by N-acetyl-L-cysteine in this combination gave practically the same protective effect. With this combination, different routes of drug administration (i.e., protectants given intraperitoneally followed by acetaldehyde given orally) also demonstrated the protective effect, but to a somewhat lesser degree than the oral route for both. To the best of our knowledge, our findings demonstrate for the first time that direct protective action against acetaldehyde toxicity and lethality can be obtained with certain naturally-occurring metabolites, namely L-ascorbic acid, L-cysteine, and thiamin, preferably in combination at reduced dose levels. Thus, our findings point the way to a possible buildup of natural protection against the chronic body insult of acetaldehyde arising from heavy drinking of alcohol and heavy smoking of cigarettes. We hasten to add, however, that such an implication from our findings must be further evaluated in animals before any extrapolation of it for human use can be considered. The last two sentences in the above paragraph require further elaboration. This can, perhaps, best be done by a survey of published reports already in the literature. These reports when pieced together support the rationale that ascorbic acid, cysteine, thiamin, and certain other sulfur compounds may be useful as long-term protectants against the chronic toxicity of acetaldehyde, which could develop with high alcohol intake and heavy cigarette smoking [4]. A brief review of these published reports is now presented in the following paragraphs. (a) L-Ascorbic acid as a possible protectant against acetaldehyde There is much literature evidence which warrants consideration of L-ascorbic acid as a possible protectant. L-ascorbic acid is known to
168 overcome the adverse symptoms [28] and the cardiac effects [29] of the disulfiram-ethanol reaction, which is presumably due to acetaldehyde [20]. Currently, the Physicians' Desk Reference of 1974 [30] recommends a high dose of ascorbic acid (1 g) for management of the severe effects of the disulfiram-ethanol reaction. Moreover, body levels of ascorbic acid are known to be depressed in alcoholics [31-34] and in heavy smokers on adequate dietary intake [35, 36]. Clinically, ascorbic acid levels chronically depressed over long time periods have been linked to cardiac disorders [37, 38], to atherosclerosis, particularly in association with smoking [39], and to deep-vein thrombosis [40], although these claims require further confirmation. Physiologically, such depressed levels could be induced by elevated acetaldehyde levels releasing catecholamines [5] which, in turn, stimulate increased utilization of ascorbic acid for their resynthesis [41]. Depressed ascorbic acid levels could also result from cigarette smoke per se which can oxidize ascorbic acid by way of a free radical intermediate [42]. Acetaldehyde in common with other aldehydes, might be involved in such an oxidation possibly by way of hemiacetal formation with the hydroxyl groups of ascorbic acid in the 2, 3 position. (b) L-cysteine as a possible protectant against acetaldehyde L-cysteine was also considered as a possible protectant for a number of reasons. As pointed out previously [1], cysteine by virtue of its (-SH) group can protect rats against the lethal pulmonary edema induced by thiourea compounds. It could act similarly to protect against the lethal pulmonary edema induced by acetaldehyde [43]. Additionally, cysteine and reduced glutathione by virtue of their (-SH) group are known to protect the phagocytoxic activity of alveolar macrophages from inhibition by cigarette smoke [44], presumably by protecting glyceraldehyde-3phosphate dehydrogenase activity [45]. This enzyme like liver aldehyde dehydrogenase is a nonspecific thiol-requiring enzyme capable of oxidizing acetaldehyde [46]. These observations further confirm the idea that cigarette smoke has oxidant activity [44] which can be prevented by sulfhydryl compounds [45] and possibly also by ascorbic acid [42]. Chemically, L-cysteine by way of its (-SH) group complexes with acetaldehyde to form L-2-methylthiazolidine-4-carboxylic acid (L-MTCA) by way of an intermediary hemiacetal
Protective Action of Ascorbic Acid and Sulfur Compounds or Schiff base, the latter being the favored intermediate [47]. As previously suggested [1], L-MTCA might then serve as a non-toxic metabolic detoxification product as well as a protectant by generating free intracellular (-SH) groups to complex with acetaldehyde more effectively [48]. Biochemically, it has already been suggested that cysteine can protect Coenzyme A against acetaldehyde inhibition by complexing preferentially with acetaldehyde [49]. Presumably, reduced glutathione, homocysteine, and N-acetyl-cysteine could also complex with acetaldehyde by virtue of their free (-SH) groups [47]. N-acetyl-L-cysteine is a well-known mucolytic agent capable of liquefying mucus and secretions associated with pulmonary disorders [50]. It is used in human therapy and is currently listed in Physicians' Desk Reference of 1974 [51]. Finally, cysteine may be important in normal cardiac function by giving rise to taurine which has been claimed to be a regulator of cell potassium in the heart [52, 53]. (c) Thiamin as a possible protectant against acetaldehyde Thiamin could also serve as a protectant against acetaldehyde for some very good reasons. Thus, as cocarboxylase (pyrophosphate) it is believed to complex normally with acetaldehyde to eventually form acetyl Coenzyme A by way of the lipoic (thioctic) acid pathway [54]. Impairment of thiamin absorption known to occur in alcoholism [55] could thus predispose to elevated acetaldehyde levels and possibly lead thereby eventually to cardiomyopathy [56]. In this connection, thiamin has long been known to be a specific therapeutic in alcoholic beriberi heart disease [56], but its value in alcoholic cardiomyopathy is less certain [56]. (d) Possible metabolic interrelationships between L-ascorbic acid, L-cysteine, and thiamin From the literature, it is also evident that certain combinations of ascorbic acid, cysteine, and thiamin could function more effectively than the individual compounds alone as protectants against the chronic toxicity of acetaldehyde. HUEPER [57], as early as 1943 in studies relating to smoking, found that dietary supplements of ascorbic acid and cystine gave rats definite protection against chronic nicotine poisoning. He then concluded that 'exogenous and possibly dietary factors may account in part for the differences in individual susceptibility to nicotine in
Protective Action of Ascorbic Acid and Sulfur Compounds man and in animals'. Metabolically, ascorbic acid, cysteine, and thiamin are known to be interrelated in ways which could enhance their individual activity in the body. For example, thiamin is known to form with cysteine a mixed disulfide which has been claimed to be a more active form of thiamin in vivo [58]. Formation of this disulfide could be effected by the cysteine-cystine redox equilibrium system which in turn is believed to be regulated by the ascorbic-dehydroascorbic acid redox system like that of glutathione [59]. This possibility is supported by reports that ascorbic acid has a thiamin-sparing action in vivo which may be due to thiamin disulfide activation in tissues by cysteine [60, 61]. Thus, the ascorbic-dehydroascorbic acid redox system may enhance thiamin activity by activating the redox systems of cysteine or glutathione to form with thiamin the more active thiamin-cysteine disulfide. Despite an element of controversy [62], the practical import of these observations is apparent from other reports that the disulfides of thiamin, notably allithiamine [63] and especially thiamin propyl disulfide [64], are more active, better absorbed, and better utilized than thiamin itself. Indeed, in alcoholic patients, thiamin propyl disulfide may correct thiamin depletion syndromes which are resistant to oral thiamine hydrochloride [65]. Finally, it is important, too, to remember that cysteine by complexing with acetaldehyde may protect the (-SH) groups of Coenzyme A and reduced lipoic acid, both of which are associated with thiamin pyrophosphate activity. Thus, combinations of ascorbic acid, cysteine, and thiamin in certain ratios could function more effectively than any of these compounds alone as protectants against acetaldehyde.
(e) Sodium metabisulfite, L-cysteic acid, and taurine as protectants against acetaldehyde From the literature, it is evident that other sulfur compounds, particularly sodium metabisulfite, L-cysteic acid, and taurine could be effective as protectants against acetaldehyde. Thus, sodium metabisulfite (Na~S20~) was recently found to protect animals (cats, mice) from acetaldehyde poisoning and alcoholic patients from the disulfiram-ethanol reaction [6@ Our findings with rats not only confirm the highly protective action of sodium metabisulfite against acetaldehyde but also reveal cysteic acid to be equally effective. Presumably, protection results from acetaldehyde complexing with the sulfite formed
169 from sodium metabisulfite or with cysteic acid. Taurine, which should presumably be equally as active, however, showed only moderate protective activity. This finding might be explained on the basis of the following speculation which also directs attention to the possible importance of taurine in alcoholic cardiomyopathy. Thus, taurine has been reported: (a) to be markedly increased in the urine of patients with acute alcoholism [67] and (b) to prevent and even reverse ventricular arrhythmias in the heart induced by epinephrine (catecholamine) by way of loss of myocardial potassium [52, 53]. Since disturbances of this nature (ventricular arrhythmias and loss of potassium) also occur in alcoholic cardiomyopathy [68], taurine could conceivably be of value in this condition. However, uptake of taufine into rat heart is slow, reaching a maximum in 3 or more days [69]. Therefore, its metabolic precursors, cysteine and cysteic acid [52], might be more effective in maintaining necessary levels of it for proper cardiac functioning. This speculation could explain the greater effectivity of cysteine and cysteic acid than taurine in our protection experiments reported herein. More experimental work is necessary, however, before any definite conclusions can be drawn.
(f) Acetaldehyde formation and binding by tissues in relation to its chronic toxicity and the implications thereof Some final discussion concerning the chronic toxicity effects of acetaldehyde is in order. Only recently have the adverse tissue effects of prolonged exposure to acetaldehyde begun to be perceived in relation to certain disease states in the cardiovascular, respiratory, and central nervous systems as well as in the aging process. Perhaps this is so because blood acetaldehyde levels when increased to measurable amounts with heavy alcohol intake remain relatively low compared with blood ethanol concentrations and show no dose nor dose-time relationships to blood ethanol concentrations [6]. The presumed explanation is that acetaldehyde is rapidly metabolized chiefly in the liver and that only a small excess passes into the blood. Blood levels thus merely reflect the total body concentration of excess free acetaldehyde not destroyed in the liver. However, three important considerations are thereby overlooked: (a) acetaldehyde formation from ethanol in tissues other than liver [70, 71], (b) the existence of acetaldehyde in blood and other tissues in two forms:
170 free and chemically-bound [72], and (c) acetaldehyde inhalation from cigarette smoke about which very little is presently known [9]. Concerning acetaldehyde formation in tissues other than liver, the enzymes catalyzing ethanol metabolism (alcohol dehydrogenase and aldehyde dehydrogenase) have been shown to be widely distributed in mammalian tissues [70, 71 ]. In the rat, these enzymes are found in a number of neural and somatic tissues known to be vulnerable to excessive ethanol in man [70]. Although the level of these enzymes in liver is highest by far, they do occur in other tissues at levels probably sufficient for local tissue metabolism of ethanol [70]. It is probable, therefore, that ethanol can be transported by the blood to tissues other than liver and metabolized therein to form the more toxic acetaldehyde. The vulnerability to acetaldehyde, as well as to ethanol, of all body tissues including liver deserves further study. Concerning the chemical-binding of acetaldehyde in blood and other tissues, evidence is rapidly accumulating of the importance of such occurrence. In blood, such binding was recently rediscovered in human erythrocytes and found to be released by precipitation or hemolysis in the presence of ethanol [73]. Serum binding of acetaldehyde was believed to be involved in the rapid incorporation of ethanol-l-14C into serum proteins [74]. Presumably, the ethanol forms acetaldehyde which then reacts with certain functional groups in proteins, e.g. free amino or free sulfhydryl groups [74]. In tissues, acetaldehyde can bind with liver and brain Coenzyme A with resultant loss in activity of the latter [75]. Acetaldehyde binding may also take part in the inhibition of certain mitochondrial functions, such as energized calcium uptake [76]. Very recently, acetaldehyde was found to inhibit cardiac microsomal protein synthesis in the guinea-pig at concentrations found in man after moderate ethanol ingestion [12]. On this basis it was speculated that such decreased protein synthesis could play a role in alcoholic cardiomyopathy [12]. F r o m the foregoing, it is obvious that evidence is increasing for the binding of acetaldehyde in blood and other tissues with possibly far reaching consequences. The potential of acetaldehyde for binding chemically with tissues has far reaching implications with respect to its chronic toxicity. Theoretically, acetaldehyde can combine with various functional groups found in proteins (amino, sulf-
Protective Action of Ascorbic Acid and Sulfur Compounds
hydryl, hydroxyl groups, etc.). Biochemically, it has been demonstrated to give a 'browning reaction' with proteins, involving amino groups, guanidyl groups, arid crosslinkage formation [77]. Immunologically, it like formaldehyde, has been found to act at low concentrations on antibodies [78, 79] and presumably also on antigens [80]. With certain antibodies (antipneumococcus serum and diphtheria antitoxin), solubilization of the antibody by the aldehydic group occurs with resultant loss of antibody protective power in the living animal [78, 79]. With certain antigens (diphtheria toxin), the aldehydic group produces toxoids by inactivating toxic groups [80]. It is important to note that this latter reaction is a slow and irreversible one [81]. It is not unreasonable therefore to suppose that acetaldehyde (and other aldehydes) can react with proteins slowly and irreversibly to result in adverse functional changes in these proteins, for example in reduced enzymatic activity or reduced metal-binding capacity. Such changes slow and irreversible at the molecular IeveI over a period of years could lead to adverse tissue changes and eventually organ dysfunction. In this context, our findings suggest that certain body compounds capable of protecting against acetaldehyde toxicity could lead to a possible build-up of natural protection against the chronic body insult of acetaldehyde arising from heavy alcohol intake and cigarette smoking. Such protection might be developed by increasing the levels of certain body metabolites reacting directly with, or involved in the enzymatic destruction of, acetaldehyde and other aldehydes. Candidates for such long-term protection are •-ascorbic acid possibly in combination with certain sulfur compounds, notably L-cysteine, L-cysteic acid, and thiamin. Further research in animals is necessary, however, before the long-term use of these compounds in high doses for human beings can be considered. Received 22 February 1975.
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Protective Action of Ascorbic Acid and Sulfur Compounds
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