The effect of dl -α-lipoic acid on heavy-metal intoxication in mice and dogs

ARCHIVES

OF

BIOCHEMISTRY

AND

BIOPHYSICS

86, 190-194 (1960)

The Effect of DL-wLipoic Acid on Heavy-Metal Intoxication in Mice and Dogs R. R. GRUNERT From the Stine Laboratory,

E. I. du Pont de Nemours Received

July

and Co., Newark,

Delaware

2, 1959

From a study of acute heavy-metal toxicity it has been found that nn-a-lipoic acid (a) is effective for the prevention and reversal of arsenic intoxication in mice and dogs; (b) effectively prevents mercury intoxication in mice provided a sufficiently large excess is used; (c) prevents gold intoxication in mice only under very specific conditions; and (d) fails to protect mice against a lethal dose of lead. 3-(6-Carboxyhexyl)-1,3-dithiolane was as effective as nn-a-lipoic acid in preventing arsenic intoxication in mice. INTRODUCTION

The selective toxicity of arsenicals for the pyruvic oxidase system has been attributed to their affinity for, and their inactivation of, the a-lipoic acid prosthetic group (l-3). The possibility that a-lipoic acid might counteract intoxication of arsenic and of other heavy metals was therefore indicated. Evidence in favor of this hypothesis was obtained with the demonstration of the protective action of cu-lipoic acid against the arsenite inhibition of the pyruvic oxidase system of Streptococcus fuecalis lOC1 (4). These experiments have been extended to a study of the effect of a-lipoic acid on heavy-metal intoxication of animals, with the favorable results included in this report. Our findings are a confirmation and extension of the results of Donatelli (5) and Cicala (6) which were published on the effective action of a-lipoic acid as an antidote for heavy-metal intoxication. MATERIALS

AND

METHODS

The mice used in all of the experiments were young adult males, of the Swiss Webster strain, maintained on Purina Lab Chow pellets and tap water. ad libitum. All compounds were administered in isotonic saline solution. Intoxication with arsenic or mercury was accomplished by the subcutaneous injection of sodium arsenite or mercuric chloride. In the studies with lead and gold, the mice received intraperitoneal injections of lead 190

diacetate [Pb(OAc)2.3HzO] and gold sodium thiosulfate, respectively. Treatment of these mice was by the alternate intraperitoneal or subcutaneous route using freshly prepared solutions of DL-a lipoic acid, DL-3-(6-carboxyhexyl)-1,2-dithiolane (an a-lipoic acid homolog), 2,3-dimercaptopropanol (BAL; British Anti-Lewisite), or thiomalic acid. Thea-lipoic acid was ground to a fine powder, dissolved in a 10% excess of fresh 5% sodium bicarbonate, and then diluted. The a-lipoic acid homolog solutions were prepared in a similar manner. The thiomalic acid solution was adjusted to pH 6.5 before final dilution. Intoxication of dogs (&ll kg. Beagles) with arsenic was accomplished by the subcutaneous injection of a neutral (pH 7.0) 5% solution of sodium arsenite. Treatment with a-lipoic acid was by the intramuscular injection of a neutral (pH 7.0) 20% solution of the a-lipoic acid. EXPERIMENTAL

RESULTS

Working from the observation that a-lipoic acid effectively prevented the arsenite inhibition of the pyruvic oxidase system of S. fueca2is (4), its effect on the toxicity of arsenite for mice was studied. The results of a number of experiments are summarized in Table I. The simultaneous administration of cr-lipoic acid, by the intraperitoneal route, to mice receiving an L&o of sodium arsenite, by the subcutaneous route, effectively prevented mortality. The degree of protection increased as the molecular ratio of cy-lipoic

HEAVY-METAL

acid to sodium arsenite was increased from one to six. Two to 3 moles a-lipoic acid/mole sodium arsenite was the minimum ratio necessary to demonstrate almost complete protection. The ability of a-lipoic acid to reverse the toxic effects of arsenic was then demonstrated. Two groups of 12 mice were given a subcutaneous injection of an LD,,,, of sodium arsenite (17 mg./kg. body weight). Thirtyseven minutes later t’he mice were near death and showed characteristic symptoms of arsenic intoxication. The first group of 12 received no furt’her treatment and served as the control. The second group of 11 mice (one had already died) was injected intraperitoneally with 177 mg. a-lipoic acid/kg. body weight (molecular ratio of oc-lipoic acid to sodium arsenit’e of six). All of the treated mice recovered completely. In contrast, the twelve control mice all died. The ability of a higher homolog of a-lipoic (3 - [6 - carboxyhexyl] - 1,2 - dithiolane), BAL, and thiomalic acid to protect arsenicintoxicated mice under the conditions used for oc-lipoic acid was studied with the results shown in Fig. 1. The curve for cw-lipoic acid is derived from t,he data in Table I and is included for comparison. Both 3-(6-carboxyhexyl) -1,2-dit’hiolane and BAL were as eff ecTABLE

I

THE PREVENTION OF ARSENITE INTOXICATION OF MICE BY THE SIMULTANEOUS ADMINISTRATION OF WLIPOIC Molecular ratio of ar-lipoic acid to Number of sodiumarsenitesa.6observa’lons

ACID MortalityC Average Range

% 0 1 2 3 4 6

11 6 7 5 4 1

98 71 32 10 21 0

% 92-100 25-100 8-67 O-33 O-42

0 a-Lipoic acid injected intraperitoneally as a freshly-prepared neutral isotonic saline solution at the time of arsenite intoxication. b Sodium arsenite injected subcutaneously at a predetermined LDIOO (17-22 mg./kg. body weight,). c The per cent mortality observed in a 5.day period with 12 young adult whit,e mice per observation.

INTOXICATION

191

tive as a-lipoic acid in protecting arseniteintoxicated mice when given by the subcutaneous route simultaneously with the intraperitoneal injection of an LD,,o of sodium arsenite. The minimum ratio necessary to demonstrate essentially complete protection was 2 moles/mole sodium arsenite. The activity of the higher homolog of cy-lipoic acid was unexpected since it had failed to protect the pyruvic oxidase system of S. faecalis lOC1 from arsenite inhibition in vitro (4). The activity of BAL was as anticipated from the studies of Pet.ers and his group (l-3). Thiomalic acid, however, failed to show any protection up to a ratio of 3 moles/mole sodium arsenite, even though it had been reported more effective than BAL for protection of arsenic-intoxicated mice (7). The value of ol-lipoic acid for the treatment of arsenic intoxication was further demonstrat’ed using t’he dog as the experimental animal. After a 24-hr. fast, three male dogs, ranging in weight from 8.6 to 11.O kg., were each given a subcutaneous injection of sodium arsenit,e at a previously determined lethal dose of 10 mg./kg. body weight. The first dog (Ko. 371) received no additional treatment and served as the negative control. The second dog (No. 390) received a simultaneous intramuscular injection of a-lipoic acid at a dose of 48 mg./kg. body weight (3 molar equivalents in relat,ion to the sodium arsenite). The third dog (so. 395) received a delayed int’ramuscular injection of a-lipoic acid (1 hr. aft’er the sodium arsenite) at the same dose as the second dog (48 mg./kg. body weight). The animals were observed for a j-day period, with the following observations. Dog ?;o. 371, which received no additional developed the characteristic treatment, symptoms of emesis, weakness, and pulmonary congestion with death occurring within 145 hr. as a result of the congestion. When the ol-lipoic acid was administered simultaneously with t’he arsenic (dog So. 390)) the symptom of emesis was of the same order of magnitude and duration as that observed in the untreated dog; however, t,here was no evidence of pulmonary congestion, only slight weakness and death did not result. Additional sympt,oms of excessive thirst and

192 IO Oc

greater than that observed in the case of Whereas 2-3 moles cr-lipoic acid/ mole sodium arsenite was the minimum ratio sufficient to demonstrate protection in arsenite-intoxicated mice, a ratio of 6-8 moles ac-lipoic acid/mole mercuric chloride was necessary to protect the mercury-intoxicated mice. By comparison (Table II), BAL was slightly more effective in preventing the toxic effects of mercuric chloride, 4-6 molar equivalents being sufficient to protect the mice. It should be noted that the lowest level of cu-lipoic acid (2 molar equivalents) increased the toxicity of the mercuric chloride. The mortality was increased to 100 % in the treated group from control values of 67 and 75 %. The increased toxicity of mercury for mice when the animals were treated with only 2-3 molar equivalents cu-lipoic acid was repeatedly seen. Kot only was the mortality increased, but the majority of deaths of such mice occurred during the first 24 hr. rather than the second 24 hr. (Table III). In separate experiments a second treatment with 3 molar equivalents Lu-lipoic acid 24 hr. after intoxication failed to protect the mice. When the entire treatment was delayed for 20 hr., 6 molar equivalents ol-lipoic acid also failed to protect the animals. When lead acetate was used as the agent arsenite.

75e z ? ;r

5 0,

i 2 5-

01 0

I MOLECULAR

L RAT,O-

Protective Sodium

3 Compwnd Arsenite

FIG. 1. The effect of or-lipoic acid (a), 3--(E ~icarboxyhexyl)-1,2-dithiolane (O), 2,3-dimercaptopropanol (X), and thiomalic acid (a) on the toxicity of sodium arsenite for mice. The curve for a-lipoic acid represents the average for all experiments.

urination (strong odor) lvere observed in the first 24 hr. and of diarrhea and weight loss in the first 48 hr. Recovery was complete in 4-5 days. When the administration of (Ylipoic acid was delayed for 1 hr. (dog No. 395), by which time emesis and diarrhea had developed, the pulmonary congestion was very mild and of short, duration, and again the dog recovered. The recovery period of 4-5 days was characterized by thirst, excessive urination (odor), diarrhea, and weight loss as observed with the second dog. Further studies were carried out with mercury, lead, and gold to determine if the protection that cr-lipoic acid afforded arsenicintoxicated animals would apply more broadly to heavy-metal intoxication. Mercuric chloride-intoxicated animals were first used. It can be seen from the data in Table II that the simultaneous administration of a-lipoic acid completely protected mice which had received a let.hal dose of mercuric chloride. The number of molar equivalents necessary for protection was, however,

TABLE A

II

OF THE ABILITY OF a-LIPOIC MERCURY AND BAL TO PREVENT INTOXICATION IN MICE

COMPARISON

Molecular ratio of protective agent to mercuric chloride”

Mortality

ACID

of mice treated withbsE

or-Lipoic acid

m 0

67:;5

2 4 6 8

100 58 8 0

BALd

%

67 17 0 0

0 Mercuric chloride injected subcutaneously at a level of 20 mg./kg. body weight. b Protective agent injected intraperitoneally at the time of mercury intoxication as a freshly prepared neutral isotonic saline solution. c The per cent mortality observed within a 12day period using 12 mice per group. d British Anti - Lewisite; 2 ,3 - dimercaptopropanol.

HEAVY-METAL

for heavy-metal intoxication, it was not possible to demonstrate a protective action for a-lipoic acid. The simultaneous administration of up to 1.5 molar equivalents cr-lipoic TABLE

III

THE EFFECT OF Low LEVELS OF WLIPOIC ON THE TIME OF DEATH OF MERCURYMICE INTOXICATED

ACID

&YX 1 2 3 4 5

Control

Treated”

6 29 6 0 1

29 11 3 1 0

0 Total number of deaths each day (four experiments of 12 mice per group) due to the subcutaneous injection of mercuric chloride at a level of 20-22 mg./kg. body weight. * Treated simultaneously with cu-lipoic acid by the intraperitoneal route at a molecular ratio to the mercuric chloride of 2 and 3. TABLE THE

0 Od Od 0.5 1.0 0.5 1.0

IV

EFFECT OF LU-LIPOIC ACID ON GOLD INTOXICATION OF MICE

Molecular ratio of LIlipoic acid to gold sodium thiosulfate”

Number of daily injections*

Single Repeated Single Single Repeated Repeated

MortalityC

% 67 67 92 loo 93 100 100

TABLE THE EFFECT DEPRESSION OF GOLD

Gold sodium thiosulfat.?

mg./kg.

Number of deathsa

Time interval after treatment

%::h” e

Days

2.8 2.1 2.4 1.3 1.4 1.3 1.3

0 Gold sodium thiosulfate injected intraperitoneally at a predetermined level of 300 mg./kg. body weight. * a-Lipoic acid injected subcutaneously at the time of gold intoxication. The single injections were given at that time. The first of the repeated injections was given at that time, the remainder at daily intervals thereaft.er to survivors. c The per cent mortality observed in a lo-day period with 12 young adult albino mice per observation. d Saline control injections. e Average time of death of the mice in each group.

193

INTOXICATION

OF or-LIPOIC

V ACID

ON THE

DUE TO A MINIMUM SODIUM

THIOSULFATE

Molecular ratio ~JL-o(lipoic acid to gold sodium thiosulfateb

body wt.

0 100 100 100 100 100

GROWTH

LETHAL DOSE IN MICE

Weight change in 7 days’ Simultaneous treatment

4%hr. delayed treatment

g.

g.

4.0 -2.9 -5.1 -1.1 4.2 5.2

-5.7 -5.4 -5.6 -6.3d

a Intraperitoneal injection of a minimum lethal dose. * Subcutaneous injection of cu-lipoic acid either at the time of gold intoxication or after a 4%hr. delay. c Average weight change per mouse using 12 mice per group with a starting weight range of 18-23 g. d Seventy-five per cent mortality observed in this group within 24 hr. following the a-lipoic acid injection. e Saline control injections.

acid to mice receiving a lethal dose of lead diacetate (300 mg./kg. body weight) failed to protect the animals. The large amount of lead diacetate necessary for the LD~oo and the limiting effect of the toxicity of Lu-lipoic acid itself (acute intraperitoneal LD60 = 200-250 mg./kg. body weight) did not permit the use of a high enough ratio to demonstrate the protection seen in the mercuric chloride-intoxicated mice. Daily injections of 0.5 molar equivalent cr-lipoic acid over an ll-day period were tried but without success. Donatelli (5) also failed to protect rabbits against

an intravenous

subacute

intoxication

with lead acetate by daily treatments with cr-lipoic acid. The higher homolog of cy-lipoic acid (3-[6-carboxyhexyll-1 ,Zdithiolane) also failed to protect the lead-intoxicated mice under comparable conditions of single and repeated administration. The need for a sufficiently high molar ratio of cy-lipoic acid to heavy metal to demonstrate a protective action was more clearly seen from the studies with gold. When gold sodium thiosulfate was injected into mice at a lethal dose (300 mg./kg. body weight) the

194

GRUNERT

simultaneous administration of cr-lipoic acid (0.5 and 1.0 molar equivalent) failed to prevent mortality (Table IV). In fact, the mortality in the treated groups increased (from 67 to 100%) and occurred sooner (approximately 24 hr.). When a minimum lethal dose of the gold compound was used (100 mg./kg. body weight) the only symptom observed was a characteristic weight depression over a 7-day interval. The simultaneous administration of 2 molar equivalents cy-lipoic acid to mice receiving this dose of gold, sodium thiosulfate (Table V) completely protected the mice and permitted a normal weight gain. When treatment was delayed 48 hr., no protection could be demonstrated. In a separate experiment BAL was twice as effective as cu-lipoic acid in preventing the weight depression due to the minimum lethal dose of gold. DISCUSSION

It is evident from the data that have been presented that arsenic intoxication of mice and dogs can be both prevented and reversed by a-lipoic acid. Donatelli (5) has made similar observations using rats. Furthermore, the ability of a-lipoic acid to prevent arsenic intoxication in mice is shared by its higher homolog, 3-(6-carboxyhexyl)-1,2-dithiolane. This observation was unexpected in the light of the in vitro findings (4), since a-lipoic acid but not 3-(6-carboxyhexyl)-l ,a-dithiolane prevented the inhibition of the pyruvic oxidase system of X. ,faecaZis lOC1 by arsenite. The general application of 1,2-dithiolane compounds to the problem of arsenic intoxication appears to be indicated. It was also possible to demonstrate that cu-lipoic acid protects mice from mercury intoxication. In contrast to the studies with arsenic, protection was dependent upon treatment with a sufficiently large excess of cy-lipoic acid. When the ratio of cu-lipoic acid to mercury was low (2: I), the toxicity was

not only not prevented but actually increased. Protection could, however, be demonstrated when the ratio was increased to 6: 1. Although not tested, frequent treatments with a low ratio of cu-lipoic acid to mercury would probably have been as effective as the high ratio by virtue of maintaining the blood level of a-lipoic acid necessary for protection. In fact, Donatelli (5), using guinea pigs, did find that the administration of an equimolar amount of cu-lipoic acid, three times daily at 4-hr. intervals for the first 4 days did protect the animals against acute mercury intoxication. In the case of gold and lead intoxication it was not possible to protect mice from their lethal effect because of the limit imposed on the use of or-lipoic acid by its own toxicity. Again the low ratio of a-lipoic acid to gold that was used increased its toxicity. When a minimum lethal dose of gold was used, and the criterion of weight depression rather than mortality was used, it was possible to get a high enough ratio of ol-lipoic acid to gold to demonstrate protection. ACKNOWLEDGMENTS The author wishes to thank H. R. Rosenberg of this laboratory for his constructive criticism arising from our many discussions. I am also indebted to D. C. Fletcher, formerly of this laboratory, for his assistance in carrying out the experiments with dogs. REFERENCES I. C., J. Cell&r Com,p. Physiol. 41, Suppl. No. 1, 113 (1953). 2. WOODS, D. D., J. Gen. Microbial. 9, 151 (1953). 3. PETERS, R. A., Symposia Sot. Exptl. Hiol. No. 3, 36 (1949). 4. GRUNERT, R. R., AND ROHDENBURG, E. L., grch. Biochem. Riophys. 86, 185 (1960). 5. DONATELLI, I,., Intern. Symposium on Thioctic Acid, Naples, 1966, p. 74. 6. CICALA, V., Intern. Symposium on Thioctic Acid, Naples, 1966, p. 255. 7. Anon., Chem. and Eng. News 32, 973 (1954). 1. GUNSALUG,