To:tco~, 1973, Vol. 13, pp. 323-331 . Perpmon Ptss+. Printed in ~twt Hrihin .
TOXICITY OF THE CARDIOTOXIC PROTEIN, FLAMMUTOXIN, ISOLATED FROM THE EDIBLE MUSHROOM FLAMMULINA VELUTIPES JUNG-Ynw Lalv, HUA-LIN Wu and Gums-YuF.x SxI Institute of Biochemistry, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China (Acceptedfor publication 16 February 1975) Ln~r, Hvn-Lnv Wu and Guelr-Yue1~1 SHI. Toxicity of the cardiotoxic protein, flammutoxin, isolated from the edible mushroom Flammulina velutipes. Toxicon 13, 323-331, 1975 .-Flammutoxin is a cardiotoxic protein which has been purified from the edible mushroom, Flammulina velutipes. The protein has strong hemolytic activity against human group O red blood cells and calcium ions at physiological concentration can partially reverse the hemolytic effect . The inhibitory effect of the toxic protein on the respiration of Ehrlich ascites tumor cells was also suppressed by calcium ions. When the toxic protein was injected i.p . into mice, it caused the writhing reaction which was partially inhibited by antihistaminics. When the toxic protein was locally injected into rat paws, it induced severe edema. JUNGYAW
Two cARDIOTOxlc proteins, volvatoxin and $ammutoxin, were isolated from the edible mushrooms, Volvariella volvaceae and Flammulina velutipes, respectively (Llrt et al., 1973 ; LIx et al., 1974) . Both toxic proteins cause : (1) changes in ECG such as depression of ST segment and inversion of T wave ; (2) local irritation such as a writhing reaction or local edema ; (3) hemolysis of human group O blood cells ; (4) inhibition of the respiration of Ehrlich ascites tumor cells . This present investigation reports in further detail on some of the biological activities of flammutoxin . MATERIALS AND METHODS Oxaloacetate, sodium pyruvate, fumarate, and alpha-ketoglutaric acid, were purchased from Sigma Chemical Company. Meperidine was obtained from China Anesthestic Drug Bureau. Glycyrrhitinic acid, promethazine ydrochloride and diphenhydramine hydrochloride were obtained from Tanabe Drug Manufacturing Company, Tokyo, Japan. Determination ojlumolytic activity To 1 ml of 4~ red blood cells suspended in 001 M phosphate buffered saline, pH 7~2, 02 ml of various concentration of flammutoxin was added. The final volume was made to 2 ml by the further addition of the above buffered saline . The reaction mixture wasincubated in a waterbath at 37° for various time periods. At predetermined times, the reaction mixture was centrifuged with an International refrigerated centrifuge at 2000 rev per min, at 4° for S min. The supernatant was measured at 540 nm with a Beckman model DU Spectrophotometer and the per cent hemolytis calculated from the hemoglobin values . Measwement of tumor cell respiration Transplantation of F.hrlich ascites tumor cells was carried out as described previously (Lnv et al., 1970). Respiratory measurements were performed manometrically with a Warburg apparatus (B. Braum Apparatebau, Melsungen, W. Germany) at 37° in a final volume of 3~2 ml . Each reaction mixture containing 2 ml of tumor cell suspension (10" cells per ml), 1 ml of phosphate buffer saline, and 0~2 ml of solution containing various amounts of flammutoxin . In controls, no fiammutoxin was added. 323
Toxrcox ~grs vor.
JUNG-YAW LIN, HUA-L1N WU and GUEY-YUEH SHI
Writhing reaction Various concentrations of flammutoxin in a constant volume of 0~2 ml were injected i.p . into Swiss albino, male mice weighing ZO f l g. The mice were put in a 2000 ml beaker and the numbers of writhing reaction were recorded for 10 min. No writhing reaction was observed if the toxic protein was administered by other than i.p . injection. The antihistaminics were injected i.p . 30 min before injection of flammutoxin . Edema ojrat paws Phosphate buffered saline (0~1 ml ; 001 M, pH 7~0) containing various amounts of flammutoxin was injected into the left paw of albino male rats weighing 250 f 15 g and the right paw was injected with 0'1 ml of the same buffer without liammutoxin . After 30 min, the rat was sacrificed, paws removed and their weights measured . Measurement ofserum histamine Flammutoxin, 400 ug per kg body weight, was injected i.p, into albino male rats weighing 250 f 15 g. After various time periods the rats were decapitated and the histamine concentrations in the serum were determined fluorometrically (HACKANSON et at., 1972). RESULTS
Hemolytic activity of ~lammutoxin When flammutoxin was incubated with a ßnal concentration of 2 ~ human group O erythrocytes, it caused complete lysis of the cells within 30 min at 37° in concentration above 20 ug per ml (Fig. 1). The hemolytic activity of flammutoxin is temperature dependent . As shown in Fig . 2, there is only 5 ~ hemolysis at 0° for 30 min incubation; however, complete lysis occurs when the incubation temperature is higher than 30° . The hemolytic action is also pH dependent (Fig. 3). Between pH 7~6 and 8~5 little hemolysis is observed . At pH values lower than 4~8, the acidity itself causes the lysis of red blood cells . 100
FIG. I . SENSITIVITY OF RED BLOOD CELLO FROM VARIOUS SPECIFB TO FLAMMUTOXIN .
Various washed red blood cells from different animals were incubated with flammutoxin at various concentrations, in 001 M phosphate buffer saline pH 72 for 30 min and the reaction was centrifuged at 800 g with an International refrigerated centrifuge . The supernatant was measured at 540 nm with a Beckman model DU spectrophotometer and the hemoglobin in the supernatant was calculated against hemoglobin which was obtained by complete lysis of red blood cells by repeating freezing and thawing. "-" Rabbit ; "-" cat ; /-/human; x-x sheep, goat, toad . TUXICON 1y75 Vol. I!
Toxicity of flammutoxin ioo
FIG . 2 . EFFECT OF TEMPERANRS ON THE HEMOLYTIC ACIMTY OF FLAMMUTOXIN (2S hg per ml). Human group O type RBC were incubated with flammutoxin in pH 7~2 phosphate buffered saline for 30 min.
pH FIG . 3. EFFECT OF pH ON THE ~EMOLYTIC ACTIVITY OF FLAMMUTOXIN. The buffers used are: pH 4~4-5~6, acetate buffer, pH 58-8, phosphate buffer . Human red blood cells were incubated with ("-") or without (x -x) flammutoxin (125 lIg per ml) at 37° for 30 min. Lysis of RHC by control solutions ( x- x) was only observed at pH values below 5~2.
Divalent cations such as calcium or strontium inhibited the hemolytic action of flammutoxin. Calcium ions, at a physiological concentration (025 mM), inhibited hemolysis about 60 ~. Strontium ions have a similar effect, but magnesium, barium or manganese ions have little or no effect on hemolysis induced by flammutoxin (Table 1). 710XICON 1975 Yd. l3
JUNG-YAW L1N, HUA-LIN WU and GUEY-YUEH SHI TABLE I . EFFECT OF DIVALENT CATIONS ON THE FIEMOLYSLg OF GROUP O HUMAN RED BLOOD CELLS BY FLAMMUTOXIN (2S wg Per ml)
Mg'* Ca' * Sr'* Ba'+ Mn'*
107 43 36 79 83
Cation wnc . 0'S InM 025 mM Hemolysis 100 104 39 39 33 3S 100 100 85 77
Red blood cells from various species of animals have different sensitivity toward fla.mmutoxin. Among the red blood cells tested, rabbit cells are the most sensitive, followed by cat, rat and human. Goat, sheep and toad red blood cells are completely resistant to flammutoxin (Fig. 1). The stability of flammutoxin to heat-treatment was studied, and it was found that the treatment of the toxic protein at temperatures higher than 60° for 30 min, caused the complete inactivation of hemolytic activity of flammutoxin. Cell swelling and inhibition of cell respiration
Flammutoxin causes the swelling of Ehrlich ascites tumor cells (Fig. 4) and the volume of treated cells is about four times larger than that of control at the concentration of 20 ltg per ml. The toxic protein is able to inhibit the respiration of Ehrlich ascites tumor cells (Fig. 5) ;
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FIO. S. EFFECT OF VARIOUS CONCENTRATION3 OF PLAMMUTOXIN ON THE RESPIRATION OF EJiRLICH ASCIIF3 TUMOR CELLS.
Flammutoxin was added at zero time into the reaction mixture . " --- " Control ; " - - - " 2~S pg per ml flammutoxin ; " - - - " S ug per ml 19ammutoxin ; " - " 10 kg per ml flammutoxin ; " --- " 20 ug per ml flammutoxin . TOXICOIV l973 V°l. I3
FIG. 4. SWELLING OF EHRLICH ASCTfES TUMOR CELL3. Bars represent 2ï ~Im. (a) Control; (b) in the presence of flammutoxin (20
lOXICON 1975 Vol. 13
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Toxicity of flammutozin
almost completely suppressing oxygen uptake at a concentration of 20 lIg per ml. Divalent calcium ions can partially reverse the inhibition caused by flammutoxin (Fig. ~, and also 150
- 100 i C O â E m c 0 u N O
FIG . G . EFFECT OF CALCIUM IONS ON THE RESPAIATION OF EHRLICH ASCITE4 TUMOR CELLS .
Both flammutoxin and calcium ions were added at zero time. "- " Control ; " - " flammutoxin 10 pg per ml plus 025 mM calcium chloride ; x -- x flammutoxin 10 ug per ml .
lime, min FIG. % . EFFECT OF VARIOUS METABOLIC INTERMEDIATES ON THE INHIBTITON OF RESPIRATION OF EHRLICFI ASCrrES TUMOR CELLS BY FLAMMUTOXIN . " --- " Control ; x-x flammutoxin 20 ug per ml ; " - " flammutoxin, 20 hg per ml plus oxaloacetate 3 " 5 mM per ml ; " - " flammutozin 20 Flg per ml plus 33 mM of any one of the following compounds (a) alpha ketoglutarate, (b) pyruvate, (c) fumarate . TOXICON 1975 Yol. l3
JUNG-YAW LIN, HUA-LiN WU and GUEY-YUEH SHI
inhibits swelling caused by flammutoxin. Some metabolic intermediates pytuvate, alphaketoglutarate, oxaloacetate and fumarate were used to study the mechanism of inhibitory effect of flammutoxin on the respiration of tumor cells. Only oxaloacetate could not reverse the inhibitory effect of $ammutoxin (Fig. ~. Local irritation Flammutoxin causes a writhing reaction when it is injected i.p. into mice. As shown in Fig. 8, no writhing was observed earlier than 60 sec, maximal writhing was observed 4 min io
e s a z
FIG . S . WItITkTING REACTION CAUSED HY PLAMIdUTOXIN .
Flammutoxin (25 ug) was administrated by i.p . injection and writhing number was Tecorded for 10 min. Each mean and standard deviation is based upon 8 observations.
after injection and the writhing reaction lasted about 12 min. The total number of writhing reactions caused by a single i.p. injection is dependent on the amount of toxic protein 20
L ao Flammutoxin,
FIG. 9. EFFECT OF VARIOUS CONCENTRATIONS OF FLAMMUTOXIN ON THE WRITFmdG REACTION.
Writhing numbers were hecorded for 10 min. Each mean and standard deviation is based upon 4 observations .
TOXICON1973 Yol. l3
Toxicity of fiammutoxin
injected (Fig. 9). The maximal number of writhing reactions is 20 at doses higher than 20 llg. Some antihistaminics and analgetics were tested for their effects on the writhing reaction caused by flammutoxin. The antihistaminics diphenhydramine, glycyrrhitinic acid and promethazine suppress the writhing reaction of flammutoxin as does the analgetic, meperidine (Fig. 10). zo
so ioo zso ioo
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FIG . LO . EFFECT' OF VARIOUS ANTHüSTAMRQIC3 AND ANALGESI7C3 ON THE WRrIiIIIYG REACI20N OF FLAMMLTCOXIIV .
The dose of 8ammutoxin used in these experiments is 25 lIg and writhing number was recorded for lOmin. "-" Glyc~rrhitinicacid ; "-" diphenhydraminehydrochloride ; x-x promethazine hydrochlonde ; "-" meperidine. Each mean and standard deviation is based upon 4 observations.
Flammutoxin also causes edema of the rat paws when the toxic protein was locally injected . The degree of edema was measured by the increase in paw weight and the results are summarized in Fig. 11 . The weight of the test paws increases more than 60 ~ at doses of flammutoxin higher than 100 leg.
FIG . ll . EFFECT OF FLAMMUTOXIN INJECTED lACALLY ON l'FIE BDEMA OF RAT PAWB . The detail of experiments is described in Materials and Methods. Each mean and standard deviation is based upon 4 observations . rOX1CON 197J Vol. J3
JUNG-YAVI' LIN, HUA-LiN V1~1 and GUEY-YUEH SHI
Flammutoxin causes the release of histamine in vivo, so that 5 nun after i.p . injection of 4001tg per kg, the histamine concentration increases to 8-fold that of the control (Fig. 12).
E 160 C
i.P . (400 pg Per Icg). Each mean and standard deviation is based upon 3 observations .
I'I(i . I2 . INCREASE OF HISTAMINE CONCENTRATION IN TkiE RAT SERUM AFTER FI,An~uroxnv
Flammutoxin isolated from Flammulina velutipes is a strong cardiotoxic protein (LIN et al., 1974). Besides its toxic effect to the heart, the protein also causes a sharp fall in blood pressure when it is injected (L,Irr et al., 1974). Respiratory failure and bleeding in the lungs was found before death. Since the toxic protein can lyse red blood cells directly without adding phospholipid, it is a direct hemolytic factor . The hemolytic action of this protein is probably different from that of some hemolytic basic proteins because the isoelectric point of flammutoxin is 4~2. The hemolytic action of flammutoxin may be due to an enzymatic reaction as judged from its dependence on temperature and pH . The red blood cells from various origins have different sensitivity toward flammutoxin which could be due to their different membrane constituents COLLEY et al., 1973). Flammutoxin causes the swelling of Ehrlich ascites tumor cells which could lead to the leakage of some ions such as calcium. Therefore, the addition of calcium ions is not only able to partially suppress the lysis of red blood cells but is also able to reverse the inhibition of respiration of the tumor cells. Since calcium ions also play an important role in myocardial contraction (NAYLER and DiINNETT, 1974), the ca.rdiotoxic action of flammutoxin could be due to its effect on the intracellular calcium concentration of the heart. Most of the metabolic intermediates tested ca.n reverse the inhibitory action of the toxic protein, except for oxaloacetic acid which could be due to its poor uptake by mitochondria (CFIAPPEL, 1968). The antihistaminics can effectively suppress the local irritation caused by flammutoxin, indicating that the toxic protein might cause the release of histamine which could lead to the fall in blood pressure, swelling of rat paws and writhing reaction . However, the anti histaminics cannot decrease the tn so of the toxic protein significantly, suggesting that flammutoxin may either cause the release of other agents besides histamine or directly attack target organs causing the death of the animals. TOXJCON 1975 Vol. 13
Toxicity of tiammutoxin
REFERENCES C~rPer., J. B. (1968) Systems for the transport of substances into mitochondria . Br . med. Bull. Z4, 150. Cota,sr, C. M., ZwMt, R. F., Ro~t.oiaerr, B. and vnx Datent, M. (1973) Lytic and non-lytic degradation of phospholipids in mammalian RBC by pure phospholipase. Biochim. biophys. Acta 307, 74. HACRANSON, R., Rotaxa>;tec, A. L. and Swr uxn, K. (] 972) Fluorometric determination of histamine with OPT. Analyt. Biochem. 47, 356. Lna, J. Y., Jt:xe, T. W., G~srr, C. C., SfiI, G. Y. and Tuxc, T. C. (1973) Isolation of a new cardiotoxic protein from the edible mushroom, Volvarklla volvaceo . Nature, Lond. 246, 524. Lna, J. Y., Lnv, Y. J., Cx>;rt, C. C., Wu, H. L., SHI, G. Y. and Jsrte, T. W. (1974) Cardiotoxic protein from edible mushrooms. Nature, Lord. 252, 235. Ltrt, J. Y., Tseartc, K. Y., Cz~x, C. C., Ltx, L. T. and Tuxc, T. C. (1970) Abrin and ricin: new antitumor substances. Nature, Lond. 227, 292. N~n,ert, W. G. and Duxxerr, J. (1974) Regulation of myocardial contraction . Adv. Cardlol. 12, 45.
TOXICON 19~3 Yol. I3