Vol. 13, No. 1 Printed in U.SA.
INFECTION AND IMMUNITY, Jan. 1976, p. 163-171 Copyright (© 1976 American Society for Microbiology
Demonstration of the Cardiotoxicity of the Thermostable Direct Hemolysin (Lethal Toxin) Produced by Vibrio parahaemolyticus TAKESHI HONDA,* KIYOTA GOSHIMA, YOSHIFUMI TAKEDA, YUKIO SUGINO, AND TOSHIO MIWATANI Department of Bacteriology and Serology, Research Institute for Microbial Diseases, Osaka University, Yamada-kami, Suita, Osaka,* and Biological Research Laboratories, Central Research Division, Takeda Chemical Industries, Ltd., Yodogawa, Osaka, Japan
Received for publication 13 August 1975
Intravenous injection of the thermostable direct hemolysin (lethal toxin) produced by Vibrio parahaemolyticus caused rapid death of rats. Studies by electroencephalography and electrocardiography showed that after intravenous injection of the toxin the electroencephalogram remained normal for quite a long time after the heart of the animals had stopped beating. Depression of intraatrial and intraventricular conduction of electrical activation, including atrioventricular block, was observed in electrocardiograms of animals injected with the toxin. The toxin was also found to be toxic to cultured mouse heart cells. When it was added to the medium, the beating rhythm of the cultured heart cells increased temporarily and then soon stopped abruptly. The effect of the toxin on cultured mouse heart cells was blocked by preincubation of the toxin with a ganglioside mixture. From these results it is concluded that the thermostable direct hemolysin (lethal toxin) has cardiotoxic activity, and thus administration of the toxin causes rapid death of animals. In the preceding paper (6), we demonstrated that the purified thermostable direct hemolysin produced by Vibrioparahaemolyticus was identical to the lethal toxin found in culture filtrates of Kanagawa phenomenon-positive strains of this organism. The toxin had a strong lethal effect on mice; intravenous injection of 5 Mg of the purified toxin killed the animals within 1 min. To explain this specific characteristic of the lethal toxin of V. parahaemolyticus, we studied possible modes of action of the toxin on experimental animals and found that the lethal toxin affects the heart of the animals so that they die soon after its injection. MATERIALS AND METHODS Isolation and purification of lethal toxin. Lethal toxin was isolated from culture filtrates of a Kanagawa phenomenon-positive strain, V. parahaemolyticus WP-1, as described previously (6) and purified by successive column chromatographies on diethylaminoethylcellulose, hydroxyapatite, and Sephadex G-200 as described previously (6). The purified toxin was demonstrated to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel disc electrophoresis and analytical ultracentrifugation and to have a molecular weight of about 42,000 (6). Assay of lethal toxicity. The lethal toxicity of the purified toxin was assayed by intravenous injection
of the toxin into 12- to 15-week-old Sprague-Dawley rats. Electroencephalography. Electroencephalograms of rats after injection of the purified toxin under anesthesia with 800 mg of urethane per kg of body weight were recorded with a Nihon-Denko polygraph. The two leads were placed in the hippocampus and the motor area, respectively. Electrocardiograms obtained with a unipolar chest lead around the apex of the heart were recorded simultaneously. Electrocardiography. Electrocardiograms of rats after injection of the purified toxin under anesthesia with 800 mg of urethane per kg of body weight were recorded with a Nihon-Denko polygraph. Three leads were used: one from electrodes in the right and left forelegs, one from electrodes in the right foreleg and left hind leg, and one from electrodes in the left foreleg and left hind leg. Culture of mouse heart cells. Mouse heart cells were cultured essentially as described previously (1, 2). Hearts were dissected from 14- to 16-day-old mouse fetuses (ddY-SLC strain, specific pathogen free). The ventricles were cut into small pieces about 0.5 to 1 mm in length and soaked for 5 min at 37 C in 5 to 10 ml of calcium- and magnesium-free phosphate-buffered saline. Then the tissue was digested for 5 min at 37 C with 4 to 6 ml of 0.06% trypsin (Difco, 1:250, dissolved in calcium- and magnesiumfree phosphate-buffered saline), using a magnetic stirrer revolving at low speed. Then the supernatant was replaced by 4 ml of 0.06% trypsin in the same
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buffer, and stirring was continued at 37 C for 8 min. This treatment with trypsin solution was repeated once or twice. The supernatants from the second to fourth trypsinization treatments were transferred to test tubes containing excess cold Eagle minimum essential medium (MEM) supplemented with 10% fetal bovine serum and centrifuged at 350 x g for 5 min. The pellets were resuspended in Eagle MEM supplemented with 10% fetal bovine serum and filtered through a Swinney filter with lens papers to remove large clumps of cells. The filtered cells were seeded into gelatin-coated petri dishes (35 mm in diameter) and incubated at 37 C in Eagle MEM supplemented with 10% fetal bovine serum under an atmosphere of 5% CO2 and 95% air. The Eagle MEM used did not contain antibiotics or phenol red. To obtain a high population of single isolated myocardial cells after cultivation for 1 day, 1 x 105 to 2 x 105 cells were seeded into each dish. To obtain a large cell cluster in the center of the dish after cultivation for 1 day (Fig. 1), 1 x 106 to 1.5 x 106 cells were seeded into each dish and the dishes were immediately subjected to gyratory shaking for 30 to 60 s (3). In this way, a large cell cluster 2 to 4 mm in diameter containing more than 105 cells was obtained after cultivation for 1 day. The beating of the cultured cells was examined with an inverted phase-contrast microscope (x 100 to 600), and the number of beats of cell clusters per
minute was counted. The temperature was kept at 36 + 1 C during the period of examination, using a thermostatically controlled chamber. The standard medium used contained the following: 116 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4, 0.9 mM NaH2PO4, 5.5 mM glucose, 10 mM N,N'-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, amino acids, vitamins, and 5% fetal bovine serum, pH 7.3. The medium was buffered with N,N'-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (one of Good's buffers). A volume of less than 0.1 ml of concentrated toxin solution that had been passed through a membrane filter (Millipore Corp.) was added to 1.9 to 2 ml of the medium with gentle shaking to give the final concentrations indicated in the figures. The toxin was dissolved in the standard medium without fetal bovine serum.
RESULTS Lethal toxicity of the toxin to rats. The lethal toxicity of the purified toxin to rats was studied. Intravenous injection of 10 to 25 ug of the toxin per rat (about 430 g of body weight) killed the animals in about 2 min, whereas a dose of 7.5 ,g of toxin per rat killed the animals in about 7 min (Table 1). After injection of 5 ,ug of toxin per rat the time of death was quite
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FIG. 1. Light micrograph of a cell cluster of cultured mouse heart cells. Mouse heart cells were cultured as described in the text. The bar represents 100 pm.
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variable, and with less than 2.5 Ag of toxin per rat no deaths were observed, even within 2 to 4 days after the injection. The symptoms of the animals before death were similar to those of mice injected with the toxin (6). Electroencephalography of rats injected with the toxin. Changes in the electroencephalograms of rats injected with the toxin were studied (Fig. 2). In this experiment a rat weighing 445 g was injected intravenously with 15 Mg of the toxin and its electroencephalogram and electrocardiogram were recorded simultane-
ously. The electroencephalograms (lines III and IV in Fig. 2) showed no significant change after intravenous injection of the toxin. On the other hand, the electrocardiogram (line II in Fig. 2) showed that the voltage increased about 13 s after the injection and that the heart stopped beating 33.5 s after the injection. The electroencephalogram remained normal for more than 50 s (until 80 s after the injection). This indicates that the effect of the toxin was primarily on the heart and that the heart stopped beating before the activity of the brain was affected. Electrocardiography of rats injected with TABLE 1. Lethal activity of the purified toxin on the toxin. For further studies on changes in the intravenous injection into ratsa electrocardiogram of rats injected with the toxin, three leads were used for electrocardiogAmt of toxin injected Survival time after injection raphy. A typical example is shown in Fig. 3A, (min SD)b (I.g of protein/rat) in which a rat weighing 448 g was injected 25.0 1.87 ± 0.22 intravenously with 7.5 mg of the toxin. The 10.0 2.15 ± 0.36 toxin was administered at 0 s. At about 15 s 7.5 7.00 ± 1.53 after the injection, the P wave became wider 5.0 180.00 ± 174.00 and higher than normal. This suggests changes 2.5 No death in intraatrial impulse conduction. Also at a Sprague-Dawley rats (12 to 15 weeks old, 433.2 about this time the voltage of QRS became ± 27.8 g) were used, and each experimental group higher and ST-T changed, suggesting changes in intraventricular impulse conduction. At consisted of five rats. b SD, Standard deviation. about 17 to 18 s after the injection, elongation of I
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the PQ interval was observed, suggesting inhibition of atrioventricular conduction. Then at about 41 s after the injection, the patterns showed change of exciting foci of the ventricle and the heart rate decreased due to reduced excitation of the heart muscle. Ventricular flutter developed about 50 s after the injection and the heart stopped beating after 148 s. Another example with a similar electrocardiogram is shown in Fig. 3B. In this experiment a rat weighing 450 g was injected with 10 ,g of the toxin. In this case, in addition to the changes observed in Fig. 3A, sinus bradycardia, due to inhibition of sinus node activity, was observed about 12 s after the injection. In addi-
tion, complete atrioventricular block developed about 23 s after the injection. Moreover, at about 27 s after the injection, marked depression of conduction of ventricular excitation was observed. Ventricular flutter developed after about 41 s, and the heart stopped beating after 127.5 s. The waves seen at 125 and 127 s after the injection were due to cramp of the legs, not to heart movements. When rats were injected with less than 2.5 ,ug of the toxin, changes such as those seen in Fig. 3A and 3B appeared. However, these changes were not severe and disappeared within a few minutes.
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from mice injected with the toxin, but it was are regular and are maintained stably for at more difficult to analyze the changes because least 5 h when the cells were incubated at 36 C the pulse of mice is more rapid than that of in the standard medium (2). When a large numrats. ber of trypsin-dispersed cells were inoculated These data indicate that the thermostable into a dish, a large cell cluster was formed in direct hemolysin (lethal toxin) produced by V. the center of the dish (Fig. 1). The cell cluster parahaemolyticus has cardiotoxic activity in an- was composed of confluent monolayers with imals. Electrocardiograms showed that the multilayered cells or cell aggregates in some chief cardiotoxic effect was depression of intra- regions. All the myocardial cells in the cell atrial and intraventricular excitation, al- cluster beat spontaneously, synchronously, and though ventricular tachycardia was also ob- regularly at 100 to 180 beats/min, and the beat served in some cases. was maintained stably for at least 24 h when Effect of toxin on the beating of cultured the cultures were incubated at 36 C in the mouse heart cells. As described previously (1- standard medium (2). The beating rate of the 3), single isolated myocardial cells from fetal cell cluster changes in response to changes of mice become attached to the surface of gelatin- temperatures and potassium and calcium concoated dishes after cultivation for 10 h. About centrations in the medium, in a manner similar 80% of the cells beat spontaneously and inde- to the normal heart (2). pendently of each other at various rates of 10 to The cardiotoxicity of the toxin was examined 260 beats/min (average, about 70 beats/min) by using cultured mouse heart cells. Figures 4 when incubated at 36 C in the standard me- through 7 show the effect of the toxin on the dium. The beating rhythms of most of the cells beating of cell clusters of mouse heart cells. In
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FIG. 4. Effect of toxin on the beating of a myocardial cell cluster cultured in vitro. Mouse heart cells were cultured as described in the text. The various amounts of toxin indicated below were added to the medium at zero time, and the number of beats of the cell cluster per minutes was counted. The ordinate shows the rate of spontaneous beating ofcell cluster as percentages of that in the absence of toxin. The concentrations oftoxin in the medium were: (A) 0.05 pg/ml; (B) 0.1 Mglml; (C) 0.2 p.gIml; and (D) 1 Mglml. Symbols: *, Normal beating; 0, weaker beating; A, no beating but no cell disintegration; A, disintegration of the cells.
the absence of toxin the cell clusters beat 105 to 180 times/min. Addition of 0.05 ug of the toxin per ml to the medium increased the beat of the cell cluster slightly, but it returned to normal within 10 min (Fig. 4A). On addition of 0.1 jig of the toxin per ml, the beating of the cell cluster was stimulated immediately after the addition of the toxin and then stopped suddenly within 1 min. A few cells in some regions of the cluster showed fibrillatory movements of high frequency (3), but these were so faint that they could not be observed under a microscope at low magnification. Then, 6 min after addition of the toxin, the beating of the cell cluster suddenly started at the normal rate again and remained unchanged during further observation (Fig. 4B). Similarly, on addition of 0.2 ,g of the toxin
ml, the beating of a cell cluster first increased, then stopped, and then started again (Fig. 4C), but the interval between the times of stopping and starting again was longer than that observed on addition of 0.1 ,g of toxin per ml. Addition of 1 ,g or more of the toxin per ml of medium also increased the beating of a cell cluster at first, but then the beating stopped suddenly and almost all myocardial cells disintegrated rapidly (Fig. 4D). Next the effect of repeated additions of the toxin to the medium on the beating of cell clusters was studied (Fig. 5). Stimulation, stopping, and recovery of the beating of cell clusters were observed each time on repeated addition of 0.1 pg of the toxin per ml. To examine the possibility that the fetal boper
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FIG. 5. Effect of repeated additions of the toxin to the medium on the beating of a myocardial cell cluster cultured in vitro. Experimental conditions were as described in the legend of Fig. 4 except that the toxin was added repeatedly, as indicated below. The concentrations of toxin added to the medium were: 0.1 pglml at arrow A and an additional 0.1 jg/ml at arrow B. Symbols are as in Fig. 4.
vine serum present in the medium is related to the recovery of beating of the cell cluster, the effect of the toxin was studied on cells cultured in the presence and absence of fetal bovine serum. The toxin had similar effects in the presence (Fig. 6A) and absence (Fig. 6B) of fetal bovine serum. Single isolated myocardial cells also lost their normal beating activity on addition of 0.1 to 0.2 .g of the toxin per ml. Some of the cells stopped beating and the others showed faint fibrillatory movements for 2 to 5 min after addition of the toxin. A detailed study on the effect of the toxin on single isolated myocardial cells is now in progress and will be published elsewhere. Effect of ganglioside on the cardiotoxicity of the toxin in cultured mouse heart cells. A ganglioside mixture (Sigma Chemical Co., type II) has been found to inhibit the hemolytic activity and lethal activity of purified toxin (6, 11). The cardiotoxicity of the toxin on cultured mouse heart cells was also blocked by preincubation of the toxin with a ganglioside mixture
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FIG. 6. Effect offetal bovine serum on the beating of a myocardial cell cluster cultured in vitro. Experinental conditions were as described in the legend of Fig. 4 except that fetal bovine serum was not added to the medium in (B). Toxin (02 pgIml) was added to the medium at zero time. Symbols are as Fig. 4.
Addition of ganglioside mixture alone did not affect the beating of cell clusters.
DISCUSSION The cardiotoxicity of streptolysin 0 has been reported. Kellner et al. (7) found that administration of solutions containing minute quanti(Fig. 7). For the results shown in Fig. 7A, toxin ties of streptolysin 0 to perfused isolated hearts that had been preincubated with ganglioside of guinea pigs, rabbits, and rats caused a signifiwas added at 21 min. The beating of a cell cant decrease in the amplitude of myocardial cluster that had been affected by the intact contraction. Halbert et al. (4) and Halpern and toxin was not affected by toxin after its preincu- Rahman (5) also reported that profound electrobation with ganglioside. Similarly, addition of cardiographic changes preceded rapid death of toxin that had been preincubated with ganglio- rabbits injected intravenously with streptolysin side at zero time did not affect the beating of a 0. Kingdon and Sword (8) demonstrated the cell cluster, whereas the cell cluster responded cardiotoxicity ofListeria monocytogenes hemolto normal toxin added at 21 min (Fig. 7B). ysin. Intravenous injection of the hemolysin
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From these observations we conclude that intravenous injection of the lethal toxin of V. parahaemolyticus primarily affects the heart, so od 7 _ _ that animals died a few minutes after its admin\T istration. Intraperitoneal injection of the toxin T also killed the animals, although this route was G+T so0less effective than the intravenous route (6). E0-4 When injected intraperitoneally the toxin probably entered the blood stream and so affected z o k sthe heart. When fetal mouse heart is dissociated into its 50 30 40 20 110 0 H cells with the aid of trypsin and component B these cells are cultivated in vitro, spontaneous E-4 beating is observed in both single isolated myocardial cells and cell clusters (1-3). Single isoIco0 A lated myocardial cells are completely separated from neighboring cells and no impulses are conT ducted from cell to cell, so each cell beats inde.o0pendently at a different rate. When cell clusters are formed, all the myocardial cells in the cluster beat spontaneously. The beating of both 1 .0 singleis isolated myocardial cells and cell clus0 10 0 regular and rhythmical in the standard 2n lo0 4a0 '0 ters TIME (MIN) medium (2). Since the cultured cells have been 7. Block of the cardiotoxic effect of the toxin separated anatomically and functionally from FI( nerves, connective tissues, and blood vessels, on cudItured mouse heart cells by a ganglioside mixexamination of the effect of the toxin on the ture. Purified toxin (10 pg) was preincubated with 8 cultured heart cells is useful for determining pg o0 f a ganglioside mixture (Sigma Chemical Co., type II) in 0.2 ml of 0.01 M tris(hydroxymethyl)- whether the toxin exerts its cardiotoxic action directly on myocardial cells. Thus, the effect of amintomethane-hydrochloride buffer (pH 7.2) at37 C for 1l 5min with gentle shaking. To dissolve the gan- the lethal toxin of V. parahaemolyticus was gliosiide mixture, it was heated in the buffer at 95 C tested on single isolated myocardial cells and for seeveral minutes before use. Toxin that had been cell clusters of these cells cultured in vitro. incut5ated with the ganglioside mixture was added at or more of the purified toxin was a concent ration ofO2 pg per ml of the medium where e added per ml of the medium, the beating of both indic,ated by an arrow (G + T). Toxin that had been incutbated without the ganglioside mixture was single isolated myocardial cells and cell clusadde d where indicated by an arrow (T). Other experi- ters stopped within 1 min, indicating that the toxin affected myocardial cells directly. On addimentlal conditions were as described in the legend of tion of more than 1 gg of the toxin per ml the Fig.. 4. Symbols are as in Fig. 4. cultured myocardial cells disintegrated, but on addition of either 0.1 or 0.2 ,ug of the toxin per ml the beating of the cell clusters started again causied serious changes in the electrocardiogra: m and death of all animals within 4 to 5 within a few minutes. The resumption of beating after an interval requires further study, but min. TIuAs paper reports the cardiotoxic activity of it may be due to inactivation of the toxin during the thermostable direct hemolysin (lethal incubation. toxira) produced by V. parahaemolyticus. IntraAlthough the results shown here indicated veno us injection of the purified toxin killed rats the direct action of the toxin on the heart, it is with in a few minutes. Electrocardiographic possible that the cardiotoxicity of the toxin is chaniges were observed shortly after administra- caused by a change of ions or pH of the blood tion of the toxin, whereas no significant electro- after toxin administration. However, this is not ence phalographic changes were observed even likely for the following reasons. (i) It takes a after the heart has stopped beating. The var- fairly long time for the toxin to affect blood cells ious changes in the electrocardiogram were or tissues and cause release of ions from them very similar to those observed with L. monocy(Sakurai, Honda, Jinguji, Arita, and Miwatogetnes hemolysin by Kingdon and Sword (8). tani, manuscript in preparation). (ii) As reDep] ression of intraatrial and intraventricular ported by Sword and Kingdon (10) and conexcit tation was the most marked effect, and the firmed by us, intravenous injection of potashear t rate and rhythm were greatly affected. sium chloride to raise the blood K+ level to 20 A
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2. Goshima, K. 1975. Further studies on preservation of the beating rhythm of myocardial cells in culture. Exp. Cell Res. 92:339-349. 3. Goshima, K. 1975. Arrhythmic movements of myocardial cells in culture and their improvement with antiarrhythmic drugs. J. Mol. Cell. Cardiol. In press. 4. Halbert, S. P., R. Bircher, and E. Dahle. 1961. The analysis of streptococcal infections. V. Cardiotoxicity of streptolysin 0 for rabbits in vivo. J. Exp. Med. 113:759-785. 5. Halpern, B. N., and S. Rahman. 1968. Studies on the cardiotoxicity of streptolysin 0. Br. J. Pharmacol. Chemother. 32:441-452. 6. Honda, T., S. Taga, T. Takeda, M. A. Hasibuan, Y. Takeda, and T. Miwatani. 1976. Identification of lethal toxin with the thermostable direct hemolysin produced by Vibrio parahaemolyticus and some physicochemical properties of the purified toxin. Infect. Immun. 13:133-139. 7. Kellner, A., A. W. Bernheimer, A. S. Carlson, and E. B. Freeman. 1956. Loss of myocardial contractility induced in isolated mammalian hearts by streptolysin 0. J. Exp. Med. 104:361-373. 8. Kingdon, G. C., and C. P. Sword. 1970. Cardiotoxic and lethal effects of Listeria monocytogenes hemolysin. Infect. Immun. 1:373-379. 9. Miwatani, T., Y. Takeda, J. Sakurai, T. Honda, and T. Takeda. 1974. Biochemical and biological properties of thermostable direct hemolysin produced by Vibrio parahaemolyticus. 10th Joint Conference of U.S.-Japan Cooperative Medical Science Program, Cholera Panel, Kyoto. 10. Sword, C. P., and G. C. Kingdon. 1971. Listeria monocytogenes toxin, p. 357-377. In S. Kadis, T. C. Montie, ACKNOWLEDGMENTS and S. J. Ajl (ed.), Microbial toxins, vol. HA. AcaWe would like to express our thanks to K. Iwama and S. demic Press Inc., New York. Nakamura for help with electroencephalographic and elec- 11. Takeda, Y., T. Takeda, T. Honda, J. Sakurai, N. Ohttrocardiographic analyses, to Y. Nimura for assistance in omo, and T. Miwatani. 1975. Inhibition of hemolytic interpretation of the electrocardiogram, and to Y. Okada activity of the thermostable direct hemolysin of Vifor critical discussion and encouragement during the course brio parahaemolyticus by ganglioside. Infect. Immun. of this study. Thanks are also due to S. Taga, M. Shimizu, 12:931-933. and Y. Sawada for technical assistance. 12. Zen-Yoji, H., Y. Kudoh, H. Igarashi, K. Ohta, and K. Fukai. 1974. Purification and identification of enteropathogenic toxins "a" and "a"' produced by Vibrio LITERATURE CITED parahaemolyticus and their biological and pathologi1. Goshima, K. 1969. Synchronized beating of and electron cal activities, p. 237-243. In T. Fujino, G. Sakaguchi, R. Sakazaki, and Y. Takeda (ed.), International symtransmission between myocardial cells mediated by heterotypic strain cells in monolayer culture. Exp. posium on Vibrio parahaemolyticus. Saikon PublishCell Res. 58:420-426. ing Co., Tokyo.
meq/liter was not lethal to normal mice. (iii) Changes in the culture conditions of mouse heart cells, such as potassium ion concentrations from 1 to 20 mM, calcium ion concentrations from 0.6 to 3.6 mM, and pH from 6.8 to 9.0, did not stop beating of cultured mouse heart cells (2). Zen-Yoji et al. (12) reported the enteropathogenicity of the hemolysin of V. parahaemolyticus. They found that 500 gg of the hemolysin gave a positive result in the rabbit ileal loop test. They also found that injection of 5 to 10 mg of the purified hemolysin into the duodenum of monkeys induced diarrhea. The cytopathic effect of the purified hemolysin on cultured FL cells has also been reported (9). However, these biological activities of the hemolysin cannot explain the rapid death of animals injected with small quantities of the purified toxin. Further studies are needed on how the cardiotoxicity of the hemolysin demonstrated in this paper is related to the biological activities reported previously. The importance of the cardiotoxicity of the hemolysin in gastroenteritis due to V. parahaemolyticus also requires further studies.
INFECTION AND IMMUNITY, Jan. 1976, p. 163-171 Copyright (© 1976 American Society for Microbiology
Demonstration of the Cardiotoxicity of the Thermostable Direct Hemolysin (Lethal Toxin) Produced by Vibrio parahaemolyticus TAKESHI HONDA,* KIYOTA GOSHIMA, YOSHIFUMI TAKEDA, YUKIO SUGINO, AND TOSHIO MIWATANI Department of Bacteriology and Serology, Research Institute for Microbial Diseases, Osaka University, Yamada-kami, Suita, Osaka,* and Biological Research Laboratories, Central Research Division, Takeda Chemical Industries, Ltd., Yodogawa, Osaka, Japan
Received for publication 13 August 1975
Intravenous injection of the thermostable direct hemolysin (lethal toxin) produced by Vibrio parahaemolyticus caused rapid death of rats. Studies by electroencephalography and electrocardiography showed that after intravenous injection of the toxin the electroencephalogram remained normal for quite a long time after the heart of the animals had stopped beating. Depression of intraatrial and intraventricular conduction of electrical activation, including atrioventricular block, was observed in electrocardiograms of animals injected with the toxin. The toxin was also found to be toxic to cultured mouse heart cells. When it was added to the medium, the beating rhythm of the cultured heart cells increased temporarily and then soon stopped abruptly. The effect of the toxin on cultured mouse heart cells was blocked by preincubation of the toxin with a ganglioside mixture. From these results it is concluded that the thermostable direct hemolysin (lethal toxin) has cardiotoxic activity, and thus administration of the toxin causes rapid death of animals. In the preceding paper (6), we demonstrated that the purified thermostable direct hemolysin produced by Vibrioparahaemolyticus was identical to the lethal toxin found in culture filtrates of Kanagawa phenomenon-positive strains of this organism. The toxin had a strong lethal effect on mice; intravenous injection of 5 Mg of the purified toxin killed the animals within 1 min. To explain this specific characteristic of the lethal toxin of V. parahaemolyticus, we studied possible modes of action of the toxin on experimental animals and found that the lethal toxin affects the heart of the animals so that they die soon after its injection. MATERIALS AND METHODS Isolation and purification of lethal toxin. Lethal toxin was isolated from culture filtrates of a Kanagawa phenomenon-positive strain, V. parahaemolyticus WP-1, as described previously (6) and purified by successive column chromatographies on diethylaminoethylcellulose, hydroxyapatite, and Sephadex G-200 as described previously (6). The purified toxin was demonstrated to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel disc electrophoresis and analytical ultracentrifugation and to have a molecular weight of about 42,000 (6). Assay of lethal toxicity. The lethal toxicity of the purified toxin was assayed by intravenous injection
of the toxin into 12- to 15-week-old Sprague-Dawley rats. Electroencephalography. Electroencephalograms of rats after injection of the purified toxin under anesthesia with 800 mg of urethane per kg of body weight were recorded with a Nihon-Denko polygraph. The two leads were placed in the hippocampus and the motor area, respectively. Electrocardiograms obtained with a unipolar chest lead around the apex of the heart were recorded simultaneously. Electrocardiography. Electrocardiograms of rats after injection of the purified toxin under anesthesia with 800 mg of urethane per kg of body weight were recorded with a Nihon-Denko polygraph. Three leads were used: one from electrodes in the right and left forelegs, one from electrodes in the right foreleg and left hind leg, and one from electrodes in the left foreleg and left hind leg. Culture of mouse heart cells. Mouse heart cells were cultured essentially as described previously (1, 2). Hearts were dissected from 14- to 16-day-old mouse fetuses (ddY-SLC strain, specific pathogen free). The ventricles were cut into small pieces about 0.5 to 1 mm in length and soaked for 5 min at 37 C in 5 to 10 ml of calcium- and magnesium-free phosphate-buffered saline. Then the tissue was digested for 5 min at 37 C with 4 to 6 ml of 0.06% trypsin (Difco, 1:250, dissolved in calcium- and magnesiumfree phosphate-buffered saline), using a magnetic stirrer revolving at low speed. Then the supernatant was replaced by 4 ml of 0.06% trypsin in the same
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buffer, and stirring was continued at 37 C for 8 min. This treatment with trypsin solution was repeated once or twice. The supernatants from the second to fourth trypsinization treatments were transferred to test tubes containing excess cold Eagle minimum essential medium (MEM) supplemented with 10% fetal bovine serum and centrifuged at 350 x g for 5 min. The pellets were resuspended in Eagle MEM supplemented with 10% fetal bovine serum and filtered through a Swinney filter with lens papers to remove large clumps of cells. The filtered cells were seeded into gelatin-coated petri dishes (35 mm in diameter) and incubated at 37 C in Eagle MEM supplemented with 10% fetal bovine serum under an atmosphere of 5% CO2 and 95% air. The Eagle MEM used did not contain antibiotics or phenol red. To obtain a high population of single isolated myocardial cells after cultivation for 1 day, 1 x 105 to 2 x 105 cells were seeded into each dish. To obtain a large cell cluster in the center of the dish after cultivation for 1 day (Fig. 1), 1 x 106 to 1.5 x 106 cells were seeded into each dish and the dishes were immediately subjected to gyratory shaking for 30 to 60 s (3). In this way, a large cell cluster 2 to 4 mm in diameter containing more than 105 cells was obtained after cultivation for 1 day. The beating of the cultured cells was examined with an inverted phase-contrast microscope (x 100 to 600), and the number of beats of cell clusters per
minute was counted. The temperature was kept at 36 + 1 C during the period of examination, using a thermostatically controlled chamber. The standard medium used contained the following: 116 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4, 0.9 mM NaH2PO4, 5.5 mM glucose, 10 mM N,N'-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, amino acids, vitamins, and 5% fetal bovine serum, pH 7.3. The medium was buffered with N,N'-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (one of Good's buffers). A volume of less than 0.1 ml of concentrated toxin solution that had been passed through a membrane filter (Millipore Corp.) was added to 1.9 to 2 ml of the medium with gentle shaking to give the final concentrations indicated in the figures. The toxin was dissolved in the standard medium without fetal bovine serum.
RESULTS Lethal toxicity of the toxin to rats. The lethal toxicity of the purified toxin to rats was studied. Intravenous injection of 10 to 25 ug of the toxin per rat (about 430 g of body weight) killed the animals in about 2 min, whereas a dose of 7.5 ,g of toxin per rat killed the animals in about 7 min (Table 1). After injection of 5 ,ug of toxin per rat the time of death was quite
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FIG. 1. Light micrograph of a cell cluster of cultured mouse heart cells. Mouse heart cells were cultured as described in the text. The bar represents 100 pm.
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variable, and with less than 2.5 Ag of toxin per rat no deaths were observed, even within 2 to 4 days after the injection. The symptoms of the animals before death were similar to those of mice injected with the toxin (6). Electroencephalography of rats injected with the toxin. Changes in the electroencephalograms of rats injected with the toxin were studied (Fig. 2). In this experiment a rat weighing 445 g was injected intravenously with 15 Mg of the toxin and its electroencephalogram and electrocardiogram were recorded simultane-
ously. The electroencephalograms (lines III and IV in Fig. 2) showed no significant change after intravenous injection of the toxin. On the other hand, the electrocardiogram (line II in Fig. 2) showed that the voltage increased about 13 s after the injection and that the heart stopped beating 33.5 s after the injection. The electroencephalogram remained normal for more than 50 s (until 80 s after the injection). This indicates that the effect of the toxin was primarily on the heart and that the heart stopped beating before the activity of the brain was affected. Electrocardiography of rats injected with TABLE 1. Lethal activity of the purified toxin on the toxin. For further studies on changes in the intravenous injection into ratsa electrocardiogram of rats injected with the toxin, three leads were used for electrocardiogAmt of toxin injected Survival time after injection raphy. A typical example is shown in Fig. 3A, (min SD)b (I.g of protein/rat) in which a rat weighing 448 g was injected 25.0 1.87 ± 0.22 intravenously with 7.5 mg of the toxin. The 10.0 2.15 ± 0.36 toxin was administered at 0 s. At about 15 s 7.5 7.00 ± 1.53 after the injection, the P wave became wider 5.0 180.00 ± 174.00 and higher than normal. This suggests changes 2.5 No death in intraatrial impulse conduction. Also at a Sprague-Dawley rats (12 to 15 weeks old, 433.2 about this time the voltage of QRS became ± 27.8 g) were used, and each experimental group higher and ST-T changed, suggesting changes in intraventricular impulse conduction. At consisted of five rats. b SD, Standard deviation. about 17 to 18 s after the injection, elongation of I
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FIG. 3. Electrocardiograms ofrats injected with toxin. Rats were injected intravenously with purified toxin at zero time, and their electrocardiograms were recorded. (A) 75 pg of toxin was injected into a rat weighing 448 g; (B) 10 pg of toxin was injected into a rat weighing 450 g. Line I, time irn seconds; line II, lead from a combination of electrodes in the right and left forelegs; line III, lead from a combination of electrodes in the right foreleg and left hind leg; line IV, lead from a combination of electrodes in the left foreleg and left hind
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the PQ interval was observed, suggesting inhibition of atrioventricular conduction. Then at about 41 s after the injection, the patterns showed change of exciting foci of the ventricle and the heart rate decreased due to reduced excitation of the heart muscle. Ventricular flutter developed about 50 s after the injection and the heart stopped beating after 148 s. Another example with a similar electrocardiogram is shown in Fig. 3B. In this experiment a rat weighing 450 g was injected with 10 ,g of the toxin. In this case, in addition to the changes observed in Fig. 3A, sinus bradycardia, due to inhibition of sinus node activity, was observed about 12 s after the injection. In addi-
tion, complete atrioventricular block developed about 23 s after the injection. Moreover, at about 27 s after the injection, marked depression of conduction of ventricular excitation was observed. Ventricular flutter developed after about 41 s, and the heart stopped beating after 127.5 s. The waves seen at 125 and 127 s after the injection were due to cramp of the legs, not to heart movements. When rats were injected with less than 2.5 ,ug of the toxin, changes such as those seen in Fig. 3A and 3B appeared. However, these changes were not severe and disappeared within a few minutes.
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from mice injected with the toxin, but it was are regular and are maintained stably for at more difficult to analyze the changes because least 5 h when the cells were incubated at 36 C the pulse of mice is more rapid than that of in the standard medium (2). When a large numrats. ber of trypsin-dispersed cells were inoculated These data indicate that the thermostable into a dish, a large cell cluster was formed in direct hemolysin (lethal toxin) produced by V. the center of the dish (Fig. 1). The cell cluster parahaemolyticus has cardiotoxic activity in an- was composed of confluent monolayers with imals. Electrocardiograms showed that the multilayered cells or cell aggregates in some chief cardiotoxic effect was depression of intra- regions. All the myocardial cells in the cell atrial and intraventricular excitation, al- cluster beat spontaneously, synchronously, and though ventricular tachycardia was also ob- regularly at 100 to 180 beats/min, and the beat served in some cases. was maintained stably for at least 24 h when Effect of toxin on the beating of cultured the cultures were incubated at 36 C in the mouse heart cells. As described previously (1- standard medium (2). The beating rate of the 3), single isolated myocardial cells from fetal cell cluster changes in response to changes of mice become attached to the surface of gelatin- temperatures and potassium and calcium concoated dishes after cultivation for 10 h. About centrations in the medium, in a manner similar 80% of the cells beat spontaneously and inde- to the normal heart (2). pendently of each other at various rates of 10 to The cardiotoxicity of the toxin was examined 260 beats/min (average, about 70 beats/min) by using cultured mouse heart cells. Figures 4 when incubated at 36 C in the standard me- through 7 show the effect of the toxin on the dium. The beating rhythms of most of the cells beating of cell clusters of mouse heart cells. In
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FIG. 4. Effect of toxin on the beating of a myocardial cell cluster cultured in vitro. Mouse heart cells were cultured as described in the text. The various amounts of toxin indicated below were added to the medium at zero time, and the number of beats of the cell cluster per minutes was counted. The ordinate shows the rate of spontaneous beating ofcell cluster as percentages of that in the absence of toxin. The concentrations oftoxin in the medium were: (A) 0.05 pg/ml; (B) 0.1 Mglml; (C) 0.2 p.gIml; and (D) 1 Mglml. Symbols: *, Normal beating; 0, weaker beating; A, no beating but no cell disintegration; A, disintegration of the cells.
the absence of toxin the cell clusters beat 105 to 180 times/min. Addition of 0.05 ug of the toxin per ml to the medium increased the beat of the cell cluster slightly, but it returned to normal within 10 min (Fig. 4A). On addition of 0.1 jig of the toxin per ml, the beating of the cell cluster was stimulated immediately after the addition of the toxin and then stopped suddenly within 1 min. A few cells in some regions of the cluster showed fibrillatory movements of high frequency (3), but these were so faint that they could not be observed under a microscope at low magnification. Then, 6 min after addition of the toxin, the beating of the cell cluster suddenly started at the normal rate again and remained unchanged during further observation (Fig. 4B). Similarly, on addition of 0.2 ,g of the toxin
ml, the beating of a cell cluster first increased, then stopped, and then started again (Fig. 4C), but the interval between the times of stopping and starting again was longer than that observed on addition of 0.1 ,g of toxin per ml. Addition of 1 ,g or more of the toxin per ml of medium also increased the beating of a cell cluster at first, but then the beating stopped suddenly and almost all myocardial cells disintegrated rapidly (Fig. 4D). Next the effect of repeated additions of the toxin to the medium on the beating of cell clusters was studied (Fig. 5). Stimulation, stopping, and recovery of the beating of cell clusters were observed each time on repeated addition of 0.1 pg of the toxin per ml. To examine the possibility that the fetal boper
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FIG. 5. Effect of repeated additions of the toxin to the medium on the beating of a myocardial cell cluster cultured in vitro. Experimental conditions were as described in the legend of Fig. 4 except that the toxin was added repeatedly, as indicated below. The concentrations of toxin added to the medium were: 0.1 pglml at arrow A and an additional 0.1 jg/ml at arrow B. Symbols are as in Fig. 4.
vine serum present in the medium is related to the recovery of beating of the cell cluster, the effect of the toxin was studied on cells cultured in the presence and absence of fetal bovine serum. The toxin had similar effects in the presence (Fig. 6A) and absence (Fig. 6B) of fetal bovine serum. Single isolated myocardial cells also lost their normal beating activity on addition of 0.1 to 0.2 .g of the toxin per ml. Some of the cells stopped beating and the others showed faint fibrillatory movements for 2 to 5 min after addition of the toxin. A detailed study on the effect of the toxin on single isolated myocardial cells is now in progress and will be published elsewhere. Effect of ganglioside on the cardiotoxicity of the toxin in cultured mouse heart cells. A ganglioside mixture (Sigma Chemical Co., type II) has been found to inhibit the hemolytic activity and lethal activity of purified toxin (6, 11). The cardiotoxicity of the toxin on cultured mouse heart cells was also blocked by preincubation of the toxin with a ganglioside mixture
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FIG. 6. Effect offetal bovine serum on the beating of a myocardial cell cluster cultured in vitro. Experinental conditions were as described in the legend of Fig. 4 except that fetal bovine serum was not added to the medium in (B). Toxin (02 pgIml) was added to the medium at zero time. Symbols are as Fig. 4.
Addition of ganglioside mixture alone did not affect the beating of cell clusters.
DISCUSSION The cardiotoxicity of streptolysin 0 has been reported. Kellner et al. (7) found that administration of solutions containing minute quanti(Fig. 7). For the results shown in Fig. 7A, toxin ties of streptolysin 0 to perfused isolated hearts that had been preincubated with ganglioside of guinea pigs, rabbits, and rats caused a signifiwas added at 21 min. The beating of a cell cant decrease in the amplitude of myocardial cluster that had been affected by the intact contraction. Halbert et al. (4) and Halpern and toxin was not affected by toxin after its preincu- Rahman (5) also reported that profound electrobation with ganglioside. Similarly, addition of cardiographic changes preceded rapid death of toxin that had been preincubated with ganglio- rabbits injected intravenously with streptolysin side at zero time did not affect the beating of a 0. Kingdon and Sword (8) demonstrated the cell cluster, whereas the cell cluster responded cardiotoxicity ofListeria monocytogenes hemolto normal toxin added at 21 min (Fig. 7B). ysin. Intravenous injection of the hemolysin
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From these observations we conclude that intravenous injection of the lethal toxin of V. parahaemolyticus primarily affects the heart, so od 7 _ _ that animals died a few minutes after its admin\T istration. Intraperitoneal injection of the toxin T also killed the animals, although this route was G+T so0less effective than the intravenous route (6). E0-4 When injected intraperitoneally the toxin probably entered the blood stream and so affected z o k sthe heart. When fetal mouse heart is dissociated into its 50 30 40 20 110 0 H cells with the aid of trypsin and component B these cells are cultivated in vitro, spontaneous E-4 beating is observed in both single isolated myocardial cells and cell clusters (1-3). Single isoIco0 A lated myocardial cells are completely separated from neighboring cells and no impulses are conT ducted from cell to cell, so each cell beats inde.o0pendently at a different rate. When cell clusters are formed, all the myocardial cells in the cluster beat spontaneously. The beating of both 1 .0 singleis isolated myocardial cells and cell clus0 10 0 regular and rhythmical in the standard 2n lo0 4a0 '0 ters TIME (MIN) medium (2). Since the cultured cells have been 7. Block of the cardiotoxic effect of the toxin separated anatomically and functionally from FI( nerves, connective tissues, and blood vessels, on cudItured mouse heart cells by a ganglioside mixexamination of the effect of the toxin on the ture. Purified toxin (10 pg) was preincubated with 8 cultured heart cells is useful for determining pg o0 f a ganglioside mixture (Sigma Chemical Co., type II) in 0.2 ml of 0.01 M tris(hydroxymethyl)- whether the toxin exerts its cardiotoxic action directly on myocardial cells. Thus, the effect of amintomethane-hydrochloride buffer (pH 7.2) at37 C for 1l 5min with gentle shaking. To dissolve the gan- the lethal toxin of V. parahaemolyticus was gliosiide mixture, it was heated in the buffer at 95 C tested on single isolated myocardial cells and for seeveral minutes before use. Toxin that had been cell clusters of these cells cultured in vitro. incut5ated with the ganglioside mixture was added at or more of the purified toxin was a concent ration ofO2 pg per ml of the medium where e added per ml of the medium, the beating of both indic,ated by an arrow (G + T). Toxin that had been incutbated without the ganglioside mixture was single isolated myocardial cells and cell clusadde d where indicated by an arrow (T). Other experi- ters stopped within 1 min, indicating that the toxin affected myocardial cells directly. On addimentlal conditions were as described in the legend of tion of more than 1 gg of the toxin per ml the Fig.. 4. Symbols are as in Fig. 4. cultured myocardial cells disintegrated, but on addition of either 0.1 or 0.2 ,ug of the toxin per ml the beating of the cell clusters started again causied serious changes in the electrocardiogra: m and death of all animals within 4 to 5 within a few minutes. The resumption of beating after an interval requires further study, but min. TIuAs paper reports the cardiotoxic activity of it may be due to inactivation of the toxin during the thermostable direct hemolysin (lethal incubation. toxira) produced by V. parahaemolyticus. IntraAlthough the results shown here indicated veno us injection of the purified toxin killed rats the direct action of the toxin on the heart, it is with in a few minutes. Electrocardiographic possible that the cardiotoxicity of the toxin is chaniges were observed shortly after administra- caused by a change of ions or pH of the blood tion of the toxin, whereas no significant electro- after toxin administration. However, this is not ence phalographic changes were observed even likely for the following reasons. (i) It takes a after the heart has stopped beating. The var- fairly long time for the toxin to affect blood cells ious changes in the electrocardiogram were or tissues and cause release of ions from them very similar to those observed with L. monocy(Sakurai, Honda, Jinguji, Arita, and Miwatogetnes hemolysin by Kingdon and Sword (8). tani, manuscript in preparation). (ii) As reDep] ression of intraatrial and intraventricular ported by Sword and Kingdon (10) and conexcit tation was the most marked effect, and the firmed by us, intravenous injection of potashear t rate and rhythm were greatly affected. sium chloride to raise the blood K+ level to 20 A
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2. Goshima, K. 1975. Further studies on preservation of the beating rhythm of myocardial cells in culture. Exp. Cell Res. 92:339-349. 3. Goshima, K. 1975. Arrhythmic movements of myocardial cells in culture and their improvement with antiarrhythmic drugs. J. Mol. Cell. Cardiol. In press. 4. Halbert, S. P., R. Bircher, and E. Dahle. 1961. The analysis of streptococcal infections. V. Cardiotoxicity of streptolysin 0 for rabbits in vivo. J. Exp. Med. 113:759-785. 5. Halpern, B. N., and S. Rahman. 1968. Studies on the cardiotoxicity of streptolysin 0. Br. J. Pharmacol. Chemother. 32:441-452. 6. Honda, T., S. Taga, T. Takeda, M. A. Hasibuan, Y. Takeda, and T. Miwatani. 1976. Identification of lethal toxin with the thermostable direct hemolysin produced by Vibrio parahaemolyticus and some physicochemical properties of the purified toxin. Infect. Immun. 13:133-139. 7. Kellner, A., A. W. Bernheimer, A. S. Carlson, and E. B. Freeman. 1956. Loss of myocardial contractility induced in isolated mammalian hearts by streptolysin 0. J. Exp. Med. 104:361-373. 8. Kingdon, G. C., and C. P. Sword. 1970. Cardiotoxic and lethal effects of Listeria monocytogenes hemolysin. Infect. Immun. 1:373-379. 9. Miwatani, T., Y. Takeda, J. Sakurai, T. Honda, and T. Takeda. 1974. Biochemical and biological properties of thermostable direct hemolysin produced by Vibrio parahaemolyticus. 10th Joint Conference of U.S.-Japan Cooperative Medical Science Program, Cholera Panel, Kyoto. 10. Sword, C. P., and G. C. Kingdon. 1971. Listeria monocytogenes toxin, p. 357-377. In S. Kadis, T. C. Montie, ACKNOWLEDGMENTS and S. J. Ajl (ed.), Microbial toxins, vol. HA. AcaWe would like to express our thanks to K. Iwama and S. demic Press Inc., New York. Nakamura for help with electroencephalographic and elec- 11. Takeda, Y., T. Takeda, T. Honda, J. Sakurai, N. Ohttrocardiographic analyses, to Y. Nimura for assistance in omo, and T. Miwatani. 1975. Inhibition of hemolytic interpretation of the electrocardiogram, and to Y. Okada activity of the thermostable direct hemolysin of Vifor critical discussion and encouragement during the course brio parahaemolyticus by ganglioside. Infect. Immun. of this study. Thanks are also due to S. Taga, M. Shimizu, 12:931-933. and Y. Sawada for technical assistance. 12. Zen-Yoji, H., Y. Kudoh, H. Igarashi, K. Ohta, and K. Fukai. 1974. Purification and identification of enteropathogenic toxins "a" and "a"' produced by Vibrio LITERATURE CITED parahaemolyticus and their biological and pathologi1. Goshima, K. 1969. Synchronized beating of and electron cal activities, p. 237-243. In T. Fujino, G. Sakaguchi, R. Sakazaki, and Y. Takeda (ed.), International symtransmission between myocardial cells mediated by heterotypic strain cells in monolayer culture. Exp. posium on Vibrio parahaemolyticus. Saikon PublishCell Res. 58:420-426. ing Co., Tokyo.
meq/liter was not lethal to normal mice. (iii) Changes in the culture conditions of mouse heart cells, such as potassium ion concentrations from 1 to 20 mM, calcium ion concentrations from 0.6 to 3.6 mM, and pH from 6.8 to 9.0, did not stop beating of cultured mouse heart cells (2). Zen-Yoji et al. (12) reported the enteropathogenicity of the hemolysin of V. parahaemolyticus. They found that 500 gg of the hemolysin gave a positive result in the rabbit ileal loop test. They also found that injection of 5 to 10 mg of the purified hemolysin into the duodenum of monkeys induced diarrhea. The cytopathic effect of the purified hemolysin on cultured FL cells has also been reported (9). However, these biological activities of the hemolysin cannot explain the rapid death of animals injected with small quantities of the purified toxin. Further studies are needed on how the cardiotoxicity of the hemolysin demonstrated in this paper is related to the biological activities reported previously. The importance of the cardiotoxicity of the hemolysin in gastroenteritis due to V. parahaemolyticus also requires further studies.
