Inhibition of release of slow-reacting substance by arsenic

EUROPEAN JOURNAL OF PHARMACOLOGY 5 (1969) 203-208. NORTH-HOLLAND PUBLISHING COMP., AMSTERDAM

INHIBITION

OF RELEASE

OF SLOW-REACTING

SUBSTANCE

BY ARSENIC

W.H.FUNDERBURK, M.FOXWELL and J.W.WARD A.H.Robins Research Laboratories, Richmond, Virginia, USA

ReceNed 19 June 1968

Accepted 17 September 1968

W.H.FUNDERBURK, M.Foxwell and J.W.WARD, Inhibition of release of slow-reacting substance by arsenic, European J. Pharmacol. 5 (1969) 203-208. The effects of arsenic, in the form of Fowler's solution, on sensitized guinea-pigs have been studied in an attempt to explain the previously reported beneficial effects of the metal in the treatment of patients with asthma. Arsenic was found to prevent the release of SRS-A and histamine in isolated perfused lungs in the presence of specific antigen. It also blocked the contraction of isolated, sensitized ileum in contact with antigen. Arsenic did not markedly inhibit the action of SRS-A, histamine or acetylcholine on isolated ileum. Some animals treated with arsenic survived anaphylactic shock whereas all untreated animals died. It was concluded that arsenic is effective in the treatment of asthmatic patients because the release of histamine and SRS-A in the lungs is blocked. Arsenic does not appear to block the union of antigen with antibody and must inhibit a later step in the reaction. The possible mechanism of action is discussed.

Arsenic Asthma Slow-reacting substance

1. INTRODUCTION Brocklehurst (1963) has shown that sensitized guinea pig and human lung tissues obtained from asthmatic patients release a substance which when placed in contact with certain isolated tissues in a water bath produces a slow sustained contraction. He called this material slow reacting substance o f anaphylaxis (SRS-A). This substance and histamine appear to be the pharmacologically active materials released during asthmatic attacks (West, 1963). Since antihistaminic drugs are of little benefit in the treatment of asthma, SRS-A may play a major role in the precipitation of such attacks. A large number of chemical compounds have been studied for possible blockade of the release of SRS-A from isolated lungs of sensitized guinea pigs. Of these

Histamine Anaphylactic shock

compounds arsenic, in the form of Fowler's solution, was found to be the most effective agent for blocking the release of this substance. The organic arsenials, oxophenarsine HC1 and 4-(4'-aminophenylazo)phenyl-arsonic acid were ineffective. It is significant that Harter and Novitch (1967) recently reported a study of the efficacy of Gay's solution in the treatment of asthma in which 13 of 18 patients in a double blind study were improved. These investigators attributed the therapeutic effects of this solution to its arsenic content which is also in the form of Fowler's solution. Other reports on the beneficial effects of Gay's solution in the treatment of this respiratory ailment have appeared in the literature (Gay and Gay, 1951 ;Waldbott, 1949) although some failures of the same treatment have also been reported (Hansen-Pruss, 1955).

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The release of SRS-A and histamine has been studied in lungs isolated from sensitized guinea pigs. SRS-A, like histamine, is usually assayed on guineapig ileum in a water bath. The two substances can be separated by using an antihistaminic drug in the bath when assaying SRS-A. The chemical structure of this substance has not been identified with certainty although a number of investigators have characterized its action on isolated ileum (Brocklehurst, 1963; Uvn~is, 1963). It produces a slow, sustained contraction and it is difficult to wash out of the tissue. Under these conditions tachyphylaxis is not encountered and usually the second application of the same amount of material produces a larger contraction than that produced by the first contact. Fowler's solution has been investigated using several immunological techniques in an attempt to examine and characterize the mechanisms by which arsenic may be involved in blockade of SRS-A release. The results of this study are reported here.

2. METHODS To sensitize guinea pigs of both sexes (Lightner Enterprize short hair) they were given 1 ml of horse serum intraperitoneally and 3 days later a second dose of 0.1 ml was administered by the same route. These animals were fed commercial guinea pig chow and cabbage and were not used for 21 days after the original sensitization procedure. To collect SRS-A and histamine from the lungs, the sensitized animals were killed with a blow on the head, the lungs were removed and the heart cut away. The lungs which were suspended in a jacketed cylinder heated with warm water were perfused through the trachea with warm Tyrode's solution (37 °C). Fluid that dripped from the lungs and cut blood vessels was collected in a funnel below. After the blood had been washed out and the perfusate was clear, 0.1 ml of horse serum was injected into the trachea. The perfusate obtained during the first 20 sec was discarded and samples of the material were then collected at 20 sec to 1 min, 1 to 3 min, 3 to 6 min and 6 to 10 min. SRS-A was assayed on isolated ileum in 25 ml aerated Tyrode's solution containing atropine sulfate ( l O - S M ) and brompheniramine maleate (3 x 10 - 8 M). Histamine was

similarly assayed without adding antihistamine drug to the bath. The effect of arsenic on the contraction of sensitized guinea-pig ileum due to direct contact with antigen [Schultz-Dale reaction (Schultz, 1910; Dale, 1913)] was studied by adding Fowler's solution and horse serum to the bath of Tyrode's solution in which atropine and brompheniramine had been omitted. Anaphylaxis was produced in sensitized guinea pigs with an intravenous injection of 1 ml of horse serum into an exposed saphenous vein. Arsenic, in the form of Fowler's solution, was given by intraperitoneal or intravenous injection 1 hr before challenge with horse serum. Arsenic was usually administered in doses of 3.8 mg/kg, a dose that has been found to be effectively and relatively non-toxic. The precipitin reaction (ring test) was used to study antigen-antibody reactions. Undiluted horse serum (0.25 ml) was placed in clean culture tubes (6 x 50 mm) and gently covered with 0.1 ml of various dilutions of serum from sensitized guinea pigs so that the two fluids formed a clear interface. Dilutions, made with physiological saline, were usually 1:1, 1:3, 1:7, 1:15, 1:31, 1:63 and 1:127. Some animals were given arsenic before sacrifice and arsenic was added to the serum at times. The tubes were refrigerated and examined for ring formation 24 hr later. Antigen-antibody reactions were further studied using the gel diffusion test. Ouchterlony plates were prepared according to methods described by Campbell et al. (1963)*. Undiluted horse serum was placed in the center well and dilutions of sensitized guineapig serum were placed in wells at the periphery. Various concentrations of arsenic were added directly to the serum before it was placed on the plates. Equal dilutions were also made of the serum without arsenic. Immunodiffusion was allowed to proceed for 24 hr at 37 °C.

3. RESULTS

3.1. Isolated lungs No attempt was made in these studies to equate * We wish to thank Donald k. Everhart, Ph.D., Medical College of Virginia, for his assistance in these studies.

ARSENIC AND SRS-A

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ARSENIC

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•

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Fig. 1. Effect of arsenic on release of SRS-A from guinea-pig lungs. Tile upper tracing is the assay of SRS-A from 2 control animals on isolated ileum. The lower tracing is the assay of 2 animals treated with 3.8 mg/kg of arsenic 1 hr before lungs were removed. Atropine and brompheniramine were added to the bath at the dot. A = 0.1 #g of ACh, No. 1 = perfusate taken from 20" to 1', N o . 2 from 1' to 3' and No. 3 from 3' to 6'. Each No. 1 signifies that perfusate from a different animal's lungs was used.

the amount of SRS-A to units as Brocklehurst (1960) has done. The amount of SRS-A obtained from isolated lungs taken from untreated animals in the first collection o f 20 sec to 1 rain after the addition o f horse serum was always very small and many times the substance could not be detected in this collection. Material collected at the 1 to 3 min interval always contained the most SRS-A; that collected at the 3 to 6 min interval contained very little active substance and the collection at the 6 to 1 0 m i n interval contained too little of the substance to be detected with this method. Histamine, or a similar substance that could be blocked with brompheniramine, was found in the 20 sec to 1 min collection but very little was usually found in the 1 to 3 min collection. Lungs taken from 12 sensitized animals were used as controls and studied for release of SRS-A and histamine. Substantial amounts of SRS-A were found in the perfusate of 11 o f these animals. There was also less histamine recovered from the animal that released no SRS-A. Arsenic (3.8 mg/kg) in the form of Fowler's solution was given intravenously to 9 sensitized guinea pigs. SRS-A was not found in the perfusate o f 8 of these animals and a slight amount was found in the 1 to 3 min collection o f the remaining animal. Arsenic

also blocked the release o f histamine. Two of three additional animals which were given 3.8 mg/kg o f the drug by i.p. injection did not have SRS-A in the perfusate of the lungs. A typical experiment o f this type is illustrated in fig. 1. 3.2. Isolated ileum The effect of arsenic on the response of isolated ileum taken from sensitized guinea pigs to the specific antigen (horse serum) was studied. In control studies sections o f ileum taken from 7 different sensitized animals all contracted in response to the addition of horse serum to the bath. After washing, none of these pieces of ileum contracted following the addition of a second 0.1 ml of horse serum. Amounts of 7.5, 11.4, 7 5 , 3 8 0 or 750/ag of arsenic were placed in the solution bathing the sensitized ileum 2 min before 0.1 ml of horse serum was added. Only the largest amount (750/lg) blocked the contraction o f the ileum. In 7 such experiments, no contraction of the ileum was observed in 5 and only very small contractions were seen in the remaining 2. Arsenic placed on other pieces o f ileum o f sensitized animals was apparently difficult to wash out. In 3 such experiments arsenic was placed in the water bath for 2 rain and following up to 6 washings, horse serum did not cause a contraction of the ileum.

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In similar studies, arsenic also blocked the Schultz-Dale reaction when the animals from which sections of ileum were taken had been treated before sacrifice. Nine sensitized animals were given 3.8 mg/kg of arsenic by i.v. injection 1 hr before they were killed and the intestines removed. Sections of ileum taken from these animals did not respond to 0.1 or 0.2 ml of horse serum placed in the bath. Up to 750/~g of arsenic placed in the Tyrode's solution that bathed the ileum did not block the action of SRS-A or acetylcholine when these materials were added to the bath. The action of histamine was slightly inhibited under these conditions.

3.3. Intact animals Anaphylactic shock was studied in 10 untreated sensitized guinea pigs which were given 1 ml of horse serum intravenously. All of these animals died within 2 to 6 min from the effects of shock. A total of 14 sensitized animals were given 3.8 mg/kg of arsenic intraperitoneally 1 hr before the administration of 1 ml of horse serum. Of these animals, 4 were dead within 5 min, 2 more were dead within 2 hr, 4 additional were dead within 24 hr, 1 more in 48 hr and 3 survived. Five additional animals were given the same amount of arsenic by intravenous injection. Following administration of 1 ml of horse serum 1 hr later,

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HOURS

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CONTROL (10)

5-

,ARSENIC IV (5)

ARSENIC IP {14)

ARSENIC IP PLUS CA GLUCANATE (5)

Fig. 2. Protective effect of arsenic on anaphylactic shock in guinea pigs. Each decrease in shading indicates time at which animals died and size of decrease indicates number of animals dying (see scale on left). Numbers in brackets show original number in groups. See text for experimental methods.

1 animal died in 7 min, 2 died within 4 hr, 1 died in 24 hr and 1 survived. Another group of 5 animals was given 2 mg/kg of arsenic on one day and 3.8 mg/kg of arsenic on the following day by i.p. injection. A total dose of 1 0 0 m g of calcium gluconate was given to each animal on both days. One hour after the last dose of arsenic the animals were challenged with 1 ml of horse serum. One animal died 48 hr later and the remaining 4 animals survived. A summary of these results is presented in fig. 2. Animals treated with arsenic before challenge with horse serum did not escape many of the marked symptoms seen in anaphylactic shock in untreated animals. During the first few minutes following the injection of the antigen they suffered severe respiratory distress and their appearances were indistinguishable from those of untreated animals. The difference was that they did not convulse and die. Animals that lived an hour or two before dying recovered from the dyspnea but had markedly swollen and hyperemic intestines at autopsy. 3.4. Antigen-antibody reactions The precipitin test (ring test) showed that arsenic did not block the complexing of antigen with antibody since a well-defined ring was frequently formed when arsenic was added to the system or when serum from sensitized guinea pigs treated with arsenic was used. Of 5 control studies, 4 were positive for ring formation at all dilutions used. The 1 control that was negative, was negative at all dilutions suggesting that the animal was not sensitized. Four animals were given 3.8 mg/kg of arsenic before blood was taken. Of these, 2 animals were positive art all dilutions; in 1 animal the undiluted serum and the 1:1 dilution was positive and all other dilutions were negative. The tests for the remaining animal were negative at all dilutions. When 0.75, 7.5 or 75/~g of arsenic was added to the guinea-pig serum, all samples were positive for ring formation. The gel diffusion tests confirmed and extended the above finding. Serum from sensitized guinea pigs in three lower peripheral wells of Ouchterlony plates were diluted with saline containing 7.5, 75, or 750/lg of arsenic. Serum placed in corresponding wells on the opposite side was diluted with equal amounts of saline and the two upper wells contained undiluted

ARSENIC AND SRS-A serum. Twenty-four hours after horse serum was added to the center well, a continuous precipitin line was found between the center well and the peripheral wells indicating that arsenic did not interfere with the antigen-antibody reaction and did not alter the antibody combining capacity.

4. DISCUSSION The results of this study suggest that arsenic has been found effective in the treatment of asthma because SRS-A and histamine release in the lungs are blocked by the drug. Arsenic apparently does not prevent the complexing of antigen with antibody but must inhibit a later step in the reaction. This is suggested by the studies of antigen-antibody reactions and the finding that sensitized animals treated previously with arsenic have marked symptoms of anaphylaxis when injected with horse serum but the response is frequently delayed and many do not die. Since no estimation of steroid levels was made in this study, it is not possible to state with certainty that arsenic did not cause stress with subsequent hormone release which could account for the partial protection in the experiments on anaphylaxis. Arsenic, however, did act locally on the sensitized ileum where steroids were not present showing that stressinduced steroid release could not be the sole mechanism of its action in these studies. The mechanism of the action of arsenic was not elucidated in these studies. Unlike some steroids, it is not effective in some other types of sensitization phenomenon; for example, it has no inhibitory~effects on experimental allergic encephalitis in guinea pigs or adjuvant arthritis in rats in other studies in this laboratory. Arsenic appears to be effective only in allergic conditions that depend upon mast cells for the pharmacologically active substances responsible for the allergic syndrome. Both SRS-A and histamine have been found to be released from mast cells in response to specific antigen (Mota and Vugman, 1956; Uvn~is and Thon, 1959). This, however, has been questioned recently by Orange, Valentine and Austen (1967), who presented data suggesting that it is the polymorphonuclear leukocyte, rather than mast cells, that plays an essential role in the release of SRS-A.

207

According to Mongar and Schild (1957) the release of histamine is the result of an enzymatic process and Brocklehurst (1960) feels that SRS-A release is also due to an enzymatic mechanism. The latter observer does not believe, however, that the same enzyme system is involved in both cases since histamine is released rapidly and SRS-A more slowly. West (1963) pointed out that sulfhydryl blocking reagents inhibit the release of histamine. Inhibition of sulfhydryl containing enzymes, then, might be the mechanism by which arsenic acts to prevent the release of histamine and SRS-A since arsenicals are known to react with sulfhydryl groups in cells (Goodman and Gilman, 1965). The finding of SRS-A in the lungs of sensitized guinea pigs and human asthmatic patients in addition to the demonstration by Brocklehurst ( 1 9 6 3 ) t h a t human bronchial rings are extremely sensitive to the action of SRS-A has provided a clue in the search for drugs that may be more useful in the treatment of asthmatic patients.

REFERENCES Brocklehurst, W.E., 1960, The release of histamine and formation of a slow-reacting substance (SRS-A) during anaphylactic shock, J. Physiol. 151,416 435. Brocklehurst, W.E., 1963, "SRS-A" The slow reacting substance of anaphylaxis, Biochem. Pharmacol. 12, 431-435. Campbell, D.H., J.S.Garvey, N.E.Cremer and D.H.Sussdorf, 1963, Methods in immunology, a laboratory text for instruction and research (W.A.Benjamin, Inc., New York) pp. 153-155. Dale, H.H., 1913, The anaphylactic reaction of plain muscle in the guinea-pig, J. Pharmacol. Exptl. Therap. 4, 167-223. Gay, F,S. and E.D.Gay, 1951, The Gay treatment of asthma, Miss. Doctor 29, 142-144. Goodman, L.S. and A.Gilman, 1965, The pharmacological basis of therapeutics, 3rd ed., (The Macmillan Co., New Yo~~r'~p. 945. Hansen-Pruss, O.C., 1955, Arsenic in the treatment of asthma, Ann. Allergy 13, 1-14. Hatter, J.G. and A.M.Novitch, 1967, An evaluation of Gay's solution in the treatment of asthma, .J. Allergy 40, 327-336. Mongar, J.L. and H.O.Schild, 1957, Effect of temperature on the anaphylactic reaction, J. Physiol. 135,320-338.

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Mota, I. and I.Vugman, 1956, Effects of anaphylactic shock and compound 48/80 on the mast cells of the guinea-pig lung, Nature (London) 177,427-429. Orange, R.P., M.D.Valentine and K.F.Austen, 1967, Release of slow-reacting substance of anaphylaxis in the rat: Polymorphonuelear leukocyte, Science 157, 318-319. Schultz, W.H., 1910, Physiological studies in anaphylaxis. 1. The reaction of smooth muscle of the guinea-pig sensitized with horse serum, J. Pharmacoi. Exptl. Therap. 1, 549 -567.

Uvn~s, B., 1963, Lipid spasmogens appearing in connection with histamine liberation, Biochem. Phaxmacol. 12, 439-443. Uvnas, B. and l.oL.Thon, 1959, Isolation of biologically intact mast cells, Expl. Cell. Res. 18,512-520. Waldbott, G.L., 1949, Symposium on treatment of long-term illnesses: New trends in the treatment of bronchial asthma, Med. Gins N. Am. 33,411-425. West, G.B., 1963, Studies on the mechanism of anaphylaxis: A possible basis for a pharmacologic approach to allergy, Clin. Pharmacol. Therap. 4 , 7 4 9 - 7 8 3 .