THE EFFECT OF ASCORBIC ACID UPON THE HEMORRHAGE PRODUCED BY BACTERIAL FILTRATE IN TRANSPLANTED TUMORS

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1 THE EFFECT OF ASCORBC ACD UPON THE HEMORRHAGE PRODUCED BY BACTERAL FLTRATE N TRANSPLANTED TUMORS HOWARD B. ANDERVONT, Sc.D.,2 AND MCHAEL B. SHMKN, M.D.3 (From the Ofice of Cancer nvestigations, United States Public Health Service, Wolcott Gibbs Memorial Laboratory, Harvard University) nterest in the effect of bacterial preparations on malignant tumors, noted over fifty years ago (l), was revived in 1931 by Gratia and Linz (2), who demonstrated hemorrhage in transplanted liposarcoma of guinea-pigs after injection of filtrate of B. COG. Various phases of this problem have been studied in this laboratory since 1932 by Andervont (3) and Shear (4). The exact nature of the effect of bacterial filtrates on transplanted tumors has not been elucidated. t is known that it resembles closely, if it is not identical with, the Shwartzman phenomenon. The most striking macroscopic and microscopic finding is that of hemorrhage in the tumor, evident about four hours after the intravenous, intraperitoneal, or subcutaneous injection of bacterial filtrate. t has been maintained, since the conclusions of Spronck (5) in 1892, that the mode of action is that of damage to the tumor capillaries by the bacterial products, with resultant bleeding and necrosis of the tumor. Apitz (6), however, noted edema of the individual tumor cells, which he believed to be independent of the hemorrhage, for while hemorrhage and edema were produced by large doses of filtrate, with smaller doses only the edema was seen. Recently, Gerber and Bernheim (7) have claimed that the effect of the filtrate on the tumor is directly on the tumor cell; their contention that " vascular alterations were not encountered " is hard to reconcile with their recognition that the outstanding features seen initially in the tumor were hemorrhage, vascular engorgement, and edema. Our experience has indicated repeatedly that the regression of transplanted tumors after bacterial filtrate treatment is present only when a gross hemorrhage is produced in the tumor, and that regression is directly proportional to the amount of hemorrhage. Thus, if only the central portion of the tumor becomes hemorrhagic, only that area regresses, while the growth of the non-hemorrhagic periphery is uninterrupted. Shwartzman (8) also states that complete regression follows only when hemorrhage appears throughout the entire tumor. t appeared significant that Harde and Kobozieff (9) and Boyland and Boyland () found that the production of hemorrhage in transplanted tumors by bacterial filtrates is accompanied by a marked reduction in the ascorbic acid content of the tumor. Since ascorbic acid is responsible for the integrity of the intercellular substance of the capillaries, it was thought that some relationship might exist between the hemorrhage and the drop in the ascorbic acid 1 Read before the American Association for Cancer Research, Richmond, Va., April 5, Senior Biologist, U. S. Public Health Service. 3 Research Fellow, National Cancer nstitute. 45 1

2 ~ t f 0 No. No. + in C.C. ET Ool / lo / lo 14 3 Y1-T 6 N TABLE 11: Effect of Neutralized Aswrbic Acid on Monoeihamlamine Aswrbate upon Hemorrhage Produced in Sarcoma 37 by B. prodigwsus FiEtrate Filtrate Ascorbic Acid (ph 6.0) and Monoethanolamine Ascorbate in Milligrams Amount Dilution No.* + t f 0 No. + f No / ABC, A and L mice with S.37 six days after grafting. *No. = total number of mice used Filtrate intraperitoneally given at same time as ascorbic acid subcutaneously. t + = definite hemorrhage; f = questionable hemorrhage; 0 = no hemorrhage.

3 HEMORRHAGE PRODUCED BY BACTERAL FLTRATE N TUMORS 453 content of the tumor. t appeared possible that the hemorrhage and resultant secondary changes in the tumor are due to the weakening of the capillary structure through the deprivation of ascorbic acid. Experiments directed toward testing this hypothesis were begun in September EXPERMENTAL Minced sarcoma 37 was inoculated into the shaved skin of the abdomen of mice. The animals were males and females of A and L strains and albino back-cross mice (ABC), and varied in age from six weeks to eight months. Six days after the grafting of the tumors, the mice were injected intraperitoneally with varying amounts and dilutions of bacterial filtrate of B. prodigiosus. t was found that 0.2 C.C. of 1/60 dilution of the filtrate in saline gave hemorrhages in almost 0 per cent of the tumors within four hours, whereas lower doses gave reactions less constantly. n all experiments the reactions were read four to five hours and twentyfour hours after injection. A definite hemorrhage is denoted in the accompanying tables by +, a questionable or very slight localized hemorrhage by -k, and tumors showing no hemorrhage are recorded as 0.. Unneutralized Ascorbic Acid: The tumor-bearing animals were divided into several groups, which were injected intraperitoneally with 0.1 C.C. of undiluted, or 0.2 C.C. of a 1/20, 1/40 or 1/60 dilution of the filtrate. A few minutes later all the controls received, subcutaneously, varying amounts (5,, 20 or 50 mg.) of ascorbic acid6 dissolved, 1 gm. per 20 c.c., in water that was free of copper and other oxidizing agents. Table, a compilation of four separate experiments of this kind, shows that the hemorrhage-producing power of the filtrate is inhibited by ascorbic acid, and that the reaction is proportional to the amount used. Thus, while 50 mg. of ascorbic acid were necessary to prevent the action of 0.1 C.C. of undiluted filtrate, the effect was obtained with mg. and less when 1/60 dilution of the filtrate was used.. Neutralized Ascorbic Acid: The ascorbic acid was neutralized to ph 6.0 by the addition of sodium hydroxide: As in the preceding experiments, varying dilutions of filtrate were given intraperitoneally, followed by the subcutaneous injection of graded doses of the neutralized ascorbic acid. Apparently ascorbic acid at ph 6.0 is somewhat unstable; if not used within a few minutes after preparation, its ability to prevent hemorrhages was lost or greatly reduced. n one series, the stable preparation for parenteral use, monoethanolamine ascorbate,7 was used. The results with the neutralized ascorbic acid and monoethanolamine ascorbate, as summarized in Table 11, are comparable with those obtained with the strongly acid compound dissolved in water. t was evident that the phenomenon was not due to the ph of the injected substance. 4 Prepared by Dr. Floyd C. Turner in May, Cebione, obtained in part gratuitously from Merck & Co. 13 Suggested by Dr. Otto A. Bessey. Cenolate, supplied gratuitously by Abbott Laboratories.

4 454 HOWARD B. ANDERVONT AND MCHAEL B. SHMKN 111. Acetic Acid Controls: To ascertain further that the acidity was not responsible for the effect observed, acetic acid of equivalent acidity to the ascorbic acid, as titrated against sodium hydroxide, was injected subcutaneously into filtrate-treated sarcoma-bearing mice. Hemorrhage was not prevented ASCORBC ACD C.C. Undiluted Ftltrotc ,. 1:20 Diluted Filtrate --**-- s 0.2. ):40.,,...*... so.z,,t;w *, N MGM. FG. 1. EFFECT OF ASCORBC ACD UPON HEMORRHAGE PRODUCED N SARCOMA B. PRODGOSUS FLTRATE (FROM TABLES 1 AND 11) 37 BY ZV. nfeuence of Reducing Power: The main properties of ascorbic acid are its acidity, its reducing power, and its antiscorbutic action. n order to ascertain the r61e of the reducing power in the phenomenon observed it was deemed desirable to study the effect of other reducing agents on the hemorrhage-producing property of bacterial filtrate. Cystein hydrochloride, neutralized to ph 6.0, was selected as being comparable. ABC mice with six-day-old intracutaneous sarcoma 37 were injected with 0.2 C.C. of a 1/20 or 1/40 dilution of the filtrate in saline intraperitoneally. A few minutes later, graded amounts of a solution containing 0 mg. of cystein hydrochloride per 1 C.C. of water, neutralized to ph 6.0 with sodium hydroxide, were injected subcutaneously. 8 Suggested by Dr. Otto A. Bessey.

5 HEMORRHAGE PRODUCED BY BACTERAL FLTRATE N TUMORS 455 TABLE 111: Effect of Cystein upon Hemorrhage in S.37 Produced by B. prodigiosus Filtrate Filtrate Cystein HC (ph 6.0) in Milligrams Amount Dilution in C.C. /lo.*l +t (f No. +lflo No. l+lflo No. 1+1flO --_ / /40 ltl-%-lt ABC, mice with S.37 six days after grafting. Filtrate intraperitoneally given at same time as cystein subcutaneously. * No. = total number of mice used. t + = definite hemorrhage; f = questionable hemorrhage; 0 = no hemorrhage. As shown in Table 11 and Figure 2, there was a marked drop in the number of hemorrhages in the tumors; whereas the controls had 93 per cent definite hemorrhages, with 30 to 40 mg. of cystein the incidence dropped to 30 per cent. The results suggest, therefore, that the reducing power of ascorbic acid lis involved in its hemorrhage-inhibiting property. V. Routes of njection: Experiments of the type described under and 1 were performed with variations in the routes of injection. The same effect was observed when both the filtrate and the ascorbic acid were injected intraperitoneally, or when the filtrate was given subcutaneously and the ascorbic acid intraperitoneally. These modes of administration proved unsatisfactory, TABLE V: Time Factor in the Effect of Ascorbic Acid upon Hemorrhage in Sarcoma 37 Produced by Bacterial Filtrate (Filtrate 0.1 C.C. undiluted, intraperitoneally ; ascorbic acid, unneutralized and at ph 6.0, 50 mg. subcutaneously) njections and Time Filtrate alone Filtrate 2 hrs. before Cevitamic acid Filtrate 1 hr. before Cevitamic acid Filtrate 1/2 hr. before Cevitamic acid Filtrate same time as Cevitamic acid Filtrate 1/2 hr. after Cevitamic acid Filtrate 1 hr. after Cevitamic acid Filtrate 2 hrs. after Cevitamic acid No. njected Hemorrhage + f l o however, because the filtrate given subcutaneously produced hemorrhages less consistently and unneutralized ascorbic acid intraperitoneally was toxic because of its acidity. The unneutralized ascorbic acid when given subcutaneously was very irritating, and caused sloughing of the skin at the injection site in 55 to 70 per cent of the animals. VZ. Time Relationship: The time relationship of the effect was studied by giving the ascorbic acid at varying intervals before and after the filtrate.

6 456 HOWARD B. ANDERVONT AND MCHAEL B. SHMKN The results, presented in Table V, show that the hemorrhage is inhibited most consistently when the ascorbic acid is given in the range of one-half hour before or after the filtrate. VZZ. Eflect Upon Crocker Sarcoma 180: n order to eliminate the possibility that the effect was limited to the tumor used, a small series of tests were made with Crocker sarcoma 180 eight days after inoculation into strain A mice. n this instance the groups treated with ascorbic acid were divided into those receiving the unneutralized acid dissolved in water, mg. per 0.1 c.c., and the compound to which sodium bicarbonate, half the weight of the FG CYSTEN N M6M :20 Dtlutcd Filtrate a 1:40 g EFFECT OF CYSTEN UPON HEMORRHAGE B. PRODGOSUS FLTRATE (FROM TABLE 111) PRODUCED N SARCOMA 37 BY cevitamic acid, was added just before injection. This procedure is recommended by Fisher and Leake (ll), and prevented visible irritative effects at the injection site. With 0.2 C.C. of 1/20 and 1/40 dilutions of prodigiosus filtrate, 0 per cent of the animals (ls/ls) had definite hemorrhages in twenty-four hours, whereas when 30 mg. of ascorbic acid was given subcutaneously with the 1/20 dilution, or 20 mg. of ascorbic acid with 1/40 dilution, only 33 per cent (8/24) had hemorrhages in the tumors.

7 HEMORRHAGE PRODUCED BY BACTERAL FLTRATE N TUMORS 457 TABLE V: Effect of Mixing Ascorbic Acid or Cystein (9H 6.0) with Bacterial Filtrate in Vitro Each mouse received intraperitoneally 0.2 C.C. Tumor Hemorrhages 1/20 dilution of filtrate mixed No. mice with 0.3 C.C. of: njected + Saline or water ph Ascorbic acid 20 mgm. Ascorbic acid 30 mgm. Ascorbic acid 40 mgm Cystein 20 mgm. Cystein 30 mgm. Cystein 40 mgm VZ. Effect Upon Lung Tumor F: Another small series of experiments was conducted with strain A mice ten days after intracutaneous inoculation with lung tumor F. This tumor, described previously (12), was the fortyninth transplant of an adenocarcinoma of the lung that was found in a strain A mouse in The tumor is still an adenocarcinoma histologically; it was resistant to filtrate up to the twenty-fifth passage, but now responds with hemorrhage, although larger doses of filtrate are required. At this time, 0.25 C.C. of the undiluted filtrate intraperitoneally produced hemorrhages in 0 per cent of the tumors (lo/lo); when, in addition, 50 mg. of ascorbic acid were given subcutaneously, 50 per cent of the animals (5/) developed hemorrhage in the tumor in twenty-four hours. X. Growth of Tumors After Treatment: Some of the animals were observed for a week after the experimental procedure. The extent of regression of the tumor was correlated with the extent of the hemorrhage, and no difference was seen between the animals which had received ascorbic acid and those which had not. f, despite the ascorbic acid, hemorrhage was present in the tumor, the hemorrhagic area regressed exactly as in the controls treated with the filtrate alone; if the ascorbic acid inhibited the hemorrhage, the tumor grew as well as in the untreated animals. X. n vitro Experiments: To observe the effect of ascorbic acid and cystein upon bacterial filtrate in vitro, 0.2 C.C. of 1/20 dilution of filtrate was mixed with graded amounts (20 to 40 mg.) of ascorbic acid or cystein neutralized to ph 6.0, so that the final volume of each injection was 0.5 C.C. As a control, water or saline brought to ph 6.0 with hydrocholoric acid was used to dilute the filtrate to 0.5 C.C. per injection. A few minutes after preparation the mixtures were injected intraperitoneally into ABC mice inoculated with sarcoma 37. As was expected from the previous observation (V), which showed that when filtrate and ascorbic acid both were injected intraperitoneally the tumor hemorrhages were inhibited, the hemorrhages were reduced to 36.7 per cent. Surprisingly, however, the stronger reducing agent, cystein, when mixed with filtrate did not lower significantly the number of hemorrhages (80 per cent). The results are summarized in Table V.

8 458 HOWARD B. ANDERVONT AND MCHAEL B. SHMKN DSCUSSON Biochemical analyses of tumors have shown that tumor tissue contains at least two reducing agents: glutathione and ascorbic acid (13). t has been suggested (14) that the function of glutathione is to protect the ascorbic acid from oxidation. Since the injection of bacterial filtrate reduces markedly (50 per cent) the ascorbic acid content of tumors, as well as of other tissues (9, lo), it is possible that the hemorrhage in tumors produced by bacterial filtrates is due to the destruction of the ascorbic acid, with resultant damage to the intercellular substance of the fragile, newly formed capillaries and hemorrhagic extravasation. Although occasional slight hemorrhages are seen in other tissues of the body after injection of bacterial filtrate, the action is significantly localized in the tumor. n this connection, it is interesting that Duran- Reynals ( 15 reports that foreign sera localize selectively in transplanted and spontaneous tumors in mice; he interprets the finding as indicating that the newly formed capillaries of tumors are more permeable than the capillaries of any normal tissue. The experiments presented here demonstrate that the effect of bacterial filtrates in producing hemorrhage in transplanted tumors can be prevented in a high percentage of cases by saturating the animals with ascorbic acid or other reducing agents, such as cystein. Addition of the mice grouped in Tables and 11, graphically represented in Fig. 1, shows that with filtrate alone, hemorrhage in the tumor occurred in 91 per cent, and that only 5 per cent were clearly negative; whereas, if the groups in which the highest concentrations of ascorbic acid were used are added, 11 per cent had definite hemorrhages and 76 per cent showed no hemorrhage. This is in agreement with the view that the primary effect of filtrate on transplanted tumors in mice is upon the blood vessels of the tumor, with secondary changes in the tumor itself. ' That actual alterations in the capillaries, -i.e., frank rupture-are not seen (7) is not surprising, since as Dalldorf (16) states, even in scurvy morphologic changes in the blood vessels have not been detected; the weakness is either in the endothelial cement or the collagenous fibers ensheathing the endothelium. t has been shown that this hemorrhage-inhibiting action of ascorbic acid is not related to the ph, but that it is dependent in some way upon its reducing property, as cystein exerted the same action. That the phenomenon is not a mere destruction of the filtrate by the reducing agent is suggested by the observation that cystein, a more powerful reducing agent than ascorbic acid, did not destroy.the hemorrhage-producing activity of the filtrate when mixed with it in vitro. t is recognized that the results of in vitro experiments of this type cannot be interpreted as indicating the reaction which takes place in the animal body. Boyland (17) has shown that ascorbic acid is selectively absorbed by tumor tissue, and the saturation of the tissues with ascorbic acid at the time of administration of bacterial filtrate may protect the integrity of the capillary structure so that hemorrhagic extravasation is prohibited. Shwartzman (18) states that ascorbic acid has no effect upon the Shwartz-

9 HEMORRHAGE PRODUCED BY BACTERAL FLTRATE N TUMORS 459 man phenomenon. f this be the case, the findings reported here can be interpreted as indicating that the modes of hemorrhage production in transplanted tumors and of the Shwartzman phenomenon in prepared skin sites, by bacterial filtrates, are not identical. SUMMARY 1. Ascorbic acid prevents the appearance of hemorrhage and resultant regression of transplanted tumors treated with bacterial filtrate. The reaction between the ascorbic acid and the filtrate is proportional to the amounts used. t is independent of the route of injection and of the ph of the ascorbic acid; it is apparently related to the reducing power of the ascorbic acid. 2. A possible mode of action of bacterial filtrate on transplanted tumors is suggested. t is postulated that bacterial filtrate, by lowering suddenly the ascorbic acid content of the tumor, weakens its fragile capillaries, with resultant hemorrhagic extravasation. BBLOGRAPHY 1. FEHLESEN: Deutsch. med. Wchnschr. 8: 553, GRATA, A., AND LNZ, R.: Compt. rend. SOC. de biol. 8: 427, ANDERVONT, H. B.: Am. J. Cancer 27: 77, ANDERVONT, H. B., AND SHEAR, M. J.: Proc. SOC. Exper. Biol. & Med. 34: 673, SHEAR, M. J.: Am. J. Cancer 25: 66, SHEAR, M. J., AND ANDERVONT, H. B.: Proc. SOC. Exper. Biol. & Med. 34: 323, SPRONCK, C. H. H.: Ann. de 1 nst. Pasteur 6: 683, APTZ, K.: Ztschr. f. Krebsforsch. 40: 50, GERBER,. E., AND BERNHEM, A..: Arch. Path. 26: 971, SHWARTZMAN, G.: Arch. Path. 21: 509, HARDE, E., AND KOBOZEFF, N.: Compt. rend. SOC. de biol. 122: 744, BOYLAND, E., AND BOYLAND, M. E.: Biochem. J. 31: 454, FSHER, B. H., ANDLEAKE, C. D.: J. A. M. A. 3: 1556, ANDERVONT, H. B.: Pub. Health Reports 52: 347, BOYLAND, E.: Biochem. J. 27: 802, BOYLAND, E.: Acta Unio nternat. contra Cancrum 3: 3, DJRAN-REYNALS, F.: Am. J. Cancer 35: 98, DALLDORF, G.: J.A. M.A. 111: 1376, BOYLAND, E.: Biochem. J. 30: 1221, SHWARTZMAN, G.: Phenomenon of Local Tissue Reactivity, P. B. Hoeber, N. Y., 1937, p. 160.