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The effects of X-irradiation on the biological activity of cottonmouth moccasin (Ancistrodon piscivorus) venom
Toxicon, 1963, Vol . 1, pp. 131-136. PaQamon Preet Ltd ., Printed in Great Britain
THE EFFECTS OF X-IRRADIATION ON THE B10I,OGICAL ACTIVITY OF COTTONMOUTH MOCCASIN (.4NCISTRODON PISCIYORUS) VENOM HERSCHEL H. FLOWERS
Pathology Division, US Army Medical Research Laboratory,
Fort Knox, Kentucky
(Received 5 July 1963) Abstract-Agkisrrodon piscivorus venom in a diltuion of 1 mg venom per 10 ml distilled water was exposed to 18,375 radf263 rad of X-irradiation to determine the effect on its local reaotivity and lethality . The precipitation patterns of antigens by immunodiffusion and immunoelectrophoresis of crude and irradiated venom was compared . The results indicate that the local reactivity of the venom was reduced 50-65 per cent . The lethal effect was markedly decreased . It took almost six times the weight of lyophilized Xirradiated venom to produce the mouse LD6° . Tmmuncelectrophoresis and immunodiffusion demonstrated little difference in the pattern of antigens of X-irradiated and normal venom . INTRODUCTION
of the family Crotalidae produce venoms which are rich sources of enzymes [f], and it is the enzymes of this venom which appear to cause severe destruction of tissue at the site of envenomation . In this manner enzymes appear to contribute to the toxicity of the whole venom. The local reactions of crude venoms make difficult their use as antigens in active immunization and in the production of antivenins . Data presented in this paper show that X-irradiated Ancistrodon piscivorus venom is less nectrotizing and less lethal than untreated venom, yet following irradiation this venom retains much of the antigenic character of the unirradiated venom. These observations suggest that irradiation of venoms may be a helpful step in the eventual production of an innocuous venom toxoid . SNAKES
MATERIALS and METHODS
The venom used was obtained from a colony of about 200 cottonmouths maintained at the United States Army Medical Research Laboratory . A glass receptacle in which the venom was collected was surrounded by crushed ice to facilitate rapid cooling of the venom (Fig. 1). Each week, venom was extracted from all snakes . After extraction and cooling the venom was lyophilized and stored at 4°C. A composite pool of lyophilized venom was then prepared from the quantity collected during a 4-month period . Venom used in this study was taken from this composite pool. Lethality of the crude venom was established by intravenous injection of approximately one hundred and fifty 18-20 gm female Swiss albino mice. The LD6o was found to be 335 mg per kg of mouse (Fig. 2). It has been demonstrated that dilute solutions of enzymes are inactivated by ionizing radiation more readily than those in the dry state or those in a concentrated solution [2j. 131
~ 32
HERSCHEL H. FLOWERS
A dilution of 1 mg venom, 10 ml distilled water was selected for exposure. After exposure, the irradiated venom solution was again lyophilized for testing purposes . For testing it was redissolved in saline in the desired concentration . Control samples not irradiated demonstrated a toxicity loss of less than 10 per cent upon being lyophilized again .
N
5
95
10
90
20
80
ó 30 á oc N
10 3 BO 50
50
30 80 90 ~ 3" 0
20 3"i
3ß
3"3
3"5 3"4 3"6 YENOM (mg/kg)
3 "7
3"B
~10
3"9
4" D
FIG. 2. LD fi p A . p(SClI'Olll.S VENOM 3'îS MG~KG
The X-ray machine employed was a North American Phillips 50 kV unit. No filter was used in order that maximum absorption of soft rays could be effected . The venom solution was exposed in 50 ml aliquots in a shallow glass dish. The depth of solution was maintained at 0~9 cm. A magnetic stirrer was employed to circulate the solution and provide
ao
0 0 S 30 x
O
1 2 3 4 6 EXPOSURE TIME FOR F~304 IN MINUTES
FIG. 3. X-IRRADIATION OF VENOM
6
FIG . ~ . COLLECTION OF VENOM .
~Ï0RIilAI YEN~II
,- ~
12Q~ S~ ~~
SAIINE COl~iROl
FIG . 4. COMPARATIVE HEMOLYSIS OF DOG RED BLOOD CELLS BY PHOSPHOLIPASE .
FIC . S. INTRADERMAL REACTIONS SEEN FROM THI ; SUBCUTANEOUS SURFACE OF RABBIT SKIN IN IECFED INTRADERMALLY WITH SERIAL DILUTIONS OF X-IRRADIATED VENOM (XV) IN COMPARISON WITH LIKE DILUTIONS OF' UNTREATED VENOM (NV) .
X-irradiation on Cottonmouth Moccasin
133
even exposure. The exposure time was 120 sec. Ferrous sulfate dosimetry was used in determining the total absorbed dose. For the 120 sec exposure the total absorbed dose was calculated to be 18,375 radf263 rad (Fig. 3). The procedure utilized for testing the irradiated and control venoms were 1 . A phospholipase activity test 2. Hemorrhagic activity test 3. Lethality tests 4. Immunoelectrophoresis and immunodiffusion The venoms of the snakes of the family Crotalidae yield a rich source of phospholipase (3] . In vivo this enzyme acts on tissue lecithin and produces lysolecithin . Lysolecithin is thought to be responsbile for the dissolution of red blood cells. To determine phospholi pase activity of the irradiated and control venom a procedure to measure in vitro hemolysis of RBC's was employed. The procedure was as follows : To supply lecithin for the production of lysolecithin an egg yolk-Tris buffer substrate was prepared . Fresh egg yolks were strained through gauze and added to an equal volume of Tris buffer (0~ 1 M pH 7~ 5 with CaCls added to 10-aM). This was thoroughly mixed, then centrifuged at 4,000 rpm for 1 to 1 1/2 hr. The supernatant was collected and bottled as substrate . Human or canine erythrocytes were washed three times in physiological saline, and a 50°.~° suspension was prepared for use in the test. Weighed quantities of venom were dissolved in Tris buffer containing CaCla. Successive dilutions were made with Tris buffer. A concentration of 1 mg venom per 100 ml buffer was adequate for venoms of high activity. Tubes were filled with 0~2 ml of substrate and 005 ml of venom. These were incubated for 45 min at 37°C. After incubating, 0~ 3 ml RBC's were added. After shaking gently to mix, it was incubated for 7 min at 37°C. On removal, 5 ml of cold saline was added to each tube. For accurate determinations it was important that each tube received the same incubation time, therefore, automatic pipetting equipment was employed to minimize the time required. Centrigufation in the cold for 10-20 min at 4,000 rpm packed the unhemolysed RBC's, and the supernatant was decanted. The supernatant was then diluted with a 1% Alconox* solution to reduce trubidity and optical density, convenient for reading on the lower half of the scale. The optical density was read at 540 um . Phospholipase activity was expressed as per cent of control value . The hemorrhagic activity was determined by utilizing the rabbit skin test as described by KONDO et al. [4] and OsAxr et al. [5] . This consisted of the normal and irradiated venom being injected intradermally into rabbits. After 24 hr the animal was sacrificed and the skin removed . The hemorrhagic areas on the underside of the skin were then compared . Differences were readily apparent and the results were photographically recorded. Lethality tests were accomplished by determining the LD6o I/V in 18-20 gm female Swiss albino mice. To demonstrate gross alteration of the antigenic character of the venom produced by X-irradiation, normal venom and X-irradiated venom were run against Polyvalent Antivenin (Crotalidae)t by agar immunodiffusion and by immunoelectrophoretic precipitation, The concentration of the venom samples was 10 mg/ml Tris buffer with CaClg. 'Alconox Detergent, Alconoz Inc., New York 3, N .Y. tPolyvalent Antivenin (Crotalidae), Wyeth Laboratories, Marietta, Pa.
l34
HERSCHEL H . FLOWERS
For immunoelectrophoresis, 1 by 3 in . miscroscope slides were covered with 2~5 to 3 ml of 1%, nutrient agar . After cooling a trough 1 ~5 mm wide and 254 cm long was cut in the agar. Three mm from the trough and on each side a circular well was cut . For immunoelectrophoresis the wells were filled with venom (005 ml instilled in each well) and the current applied for 2 hr at 150 V, 2 mA f0~6 mA ;cm gel), using barbital buffer (0~5 m pH 8~6) at 22 °C . After electrophoresis had separated the venom fractions, 0~ 15 ml of the polyvalent antivenin was instilled in the trough . Lines of precipitation appeared in the agar after 24 to 48 hr of incubation at 22°C . The slides were washed for 48 hr in NaCI to remove non-precipitated protein and then stained with amídoschwarz stain (1 °,~ in 7°.~ acetic acid) . The precipitation lines of the normal venom-antivenin reaction and the Xirradiated venom-antivenin reaction could then be compared and photographed . Immunodiffusion was accomplished in a similar manner, with the exception than an electrical current was not applied . The venom and antivenin were placed in individual wells in petri dishes filled with 15 ml agar and incubated at room temperature . Precipitation lines appeared after 18 to 24 hr, but precipitation was not complete until after 72 to 96 hr . At this time the petri dishes were photographed . Staining was unnecessary in this procedure because the lines of precipitation were very easily distinguished by eye . RESULTS Phospholipase was inactivated 95-100 per cent by radiation (Fig . 4) . The hemorrhagc skin test in rabbits demonstrated a decrease in the local effect of the X-irradiated venom as compared with normal venom (Fig. 5) . Both X-irradiated and normal venom in five dilutions from 1 :10 to I :160 in Tris bufferwereinjected intradermally . Only the higher concentrations of X-irradiated venom caused hemorrhage while all dilutions of normal venom caused hemorrhage. Immunodiffusion indicated some difference in the pattern of protein precipitation when tested against the polyvalent antivenin (Figs . 6 and 7) . Normal venom exhibited one large distinct band of precipitation with two to four smaller, less distinct bands behind the large one . The X-irradiated venom exhibited the same distinct large band, however, only one to three of the smaller bands were evident . These were less distinct than those demonstrated with the normal venom . Immunoelectrophoresis demonstrated much the same differences seen in the Immunodiffusion slides . The large band of precipitation was present in both the normal and Xirradiated venom . The X-irradiated venom exhibited fewer and more diffuse precipitation of the smaller bands . Perhaps the most significant alteration of venom affected by the X-irradiation was a decrease in lethality . After irradiation, 20 mg venom per kg mouse was required to produce the LDS° ; this is a sixfold decrease in lethality. This procedure was repeated numerous times with different samples of X-irradiated venom . Results ranged consistently from 198-205 mg/kg mouse . DISCUSSION Although studies have been accomplished concerning the effects of ionizing radiation on enzymes present in venom, to the best of the author's knowledge, only one instance is recorded in which whole venom was exposed to X-irradiatio~i . DUDLEY JACKSON [6J, in 1928, placed 45 mg of Crotalus atrox venom under l ;'2 in . of beef and exposed it to X-ray
FIG. Ó. COMPARATIVE PRECIPITATION BANDS FORMED IN AGAR BY X-IRRADIATED VENOM (XV) AND BY UNTREATED VENOM (NV) AGAINST POLYVALENT ANTIVENIN CROTALIDAE (PAC).
FIG . % . COMPARATIVE IMMUNOELECI'ROPHORETIC PATTERNS OF PRECIPITATION FORMED BY X IRRADIATED VENOM AND BY UNTREATED VENOM AGAINST CENTRALLY LOCATED POLYVALENT ANTIVENIN CROTALIDAE .
X-irradiation on Cottonmouth Moccasin
135
for 2 min. He then injected this material into two dogs. Both animals suffered severe local reactions and subsequent sloughing of tissue but did not die. There was no dosimetry reported with this exposure, nor can one be certain of the exact concentration of the venom injected; however, it is most likely that the concentration was much greater than the solutions used here. The present study demonstrates that the exposure of cottonmouth moccasin venom to X-irradiation decreases its local reactivity and lethal effect. These studies suggest that reduced local reactivity and lethal effect of this venom are accomplished with little change in the immunodiffusion and immunoelectrophoretic pattern of antigens. Although phospholipase was inactivated almost completely, the proteolytic enzymes remained capable of causing some hemorrhage and necrosis when injected intradermally, however, the activity of these enzymes was markedly reduced following irradiation. Thus, certain enzymes are totally inactivated while others appear to be only slightly effected . A need continues to exist for the development ofmethods useful in altering the lethality of venoms but which do not alter the antigenic character of the proteins. While several chemical agents have been employed to inhibit or minimize the local reactivity of the venom, none have been entirely successful in eliminating the lethal factors. Evidence has been presented which tends to support the belief that venoms contain, as yet unidentified, lethal factors which are not enzymic in character [7-9] . It appears possible to significantly inactivate this factor or factors by irradiation with little change in theimmunodiffusion and immuno-electrophoretic patterns of antigens. It seems improbable that the particular combination of irradiation dose and venom dilution used in this investigation represents conditions optimal for inactivation of toxicity with minimal alteration of antigenicity of the venom. It is indeed possible that other doses and dilutions may yield even greater inactivation with less effect on the antigenic character. For many years one of the greatest problems confronting researchers attempting to produce an effective antivenin to crotalid venoms has been that of the hemorrhage and local necrosis produced by the venom when injected into tissues. This has made difficult the production of antivenins of high titer and it has been an impediment to active immunization with crotalid venoms . It is, therefore, of importance that the use of ionizing radiation in the attenuation of venoms be further investigated. SUMMARY
1 . Exposure of a solution of 1 mg venom/10 ml distilled water for 120 sec (18,375 rad X263 rad) has yielded significant alterations in toxicity and enzyme activity. 2. The phospholipase has been inactivated 95-100 per cent . 3. Hemorrhagic and local nectrotizing action of the venom has been minimized. 4. It takes six times the weight of lyophilized X-irradiated venom to provide the mouse LDso as of normal venom. 5. Immunodiffusion and immunoelectrophoresis indicate little change in the precipitation pattern of antigens . Acknowledgements-The author is indebted to H. Bxews~e of the School of Medicine, University of Florida, for the development of the phospholipase determination procedure; and to Captain Goaoox MSC, Radiobiology Division, US Army Medical Research Laboratory, Fort Knox, Kentucky, for his technical assistance in the radiological aspects of this project. LODDE,
136
HERSCHEL H. FLOWERS
ZELLER, E. A.,
Advanc Etzymol. S,
REFERENCES 459, 1948 .
Parasitology, 36, 110, 1944 . Mvesncx, K., The Enzymes 4, pt . A. Academic
LEA, D. E., SMrrx, K. M., HOMES, B. and MAAKMAN, R., $LOTTA, K. H., BOYER, P. D., LARDY, H. and 1960.
[4] [5] [6] [~l [gl [9]
Koxno, H., Korroo, S., IKEZAWA, H. and MURATA, R.,
OFISAKA, H., IKEZAWA, H., KoNno, H., KONDO, S. and
Jap. J. Med. Sci. 13, 43, 1906. Ucxmn, N., Brit . J. erp. Path ., 41, 47,
W. T., J. Amer . vet., med. Ass., June 1928 . YANG, C. C., J. Formosan med. Ass., 59, 1326, 1960 . SLOTTA, K., FRANENKEL-CONRAT, H., Nature, Lond., 142, 213, 1938. GONCALVE3, J. M., L. G. Yieria Anal. Acad. Brasil. Cienc., 22, 140, 1950. JACKSON, D., HARRLSON,
Press.
1960 .
THE EFFECTS OF X-IRRADIATION ON THE B10I,OGICAL ACTIVITY OF COTTONMOUTH MOCCASIN (.4NCISTRODON PISCIYORUS) VENOM HERSCHEL H. FLOWERS
Pathology Division, US Army Medical Research Laboratory,
Fort Knox, Kentucky
(Received 5 July 1963) Abstract-Agkisrrodon piscivorus venom in a diltuion of 1 mg venom per 10 ml distilled water was exposed to 18,375 radf263 rad of X-irradiation to determine the effect on its local reaotivity and lethality . The precipitation patterns of antigens by immunodiffusion and immunoelectrophoresis of crude and irradiated venom was compared . The results indicate that the local reactivity of the venom was reduced 50-65 per cent . The lethal effect was markedly decreased . It took almost six times the weight of lyophilized Xirradiated venom to produce the mouse LD6° . Tmmuncelectrophoresis and immunodiffusion demonstrated little difference in the pattern of antigens of X-irradiated and normal venom . INTRODUCTION
of the family Crotalidae produce venoms which are rich sources of enzymes [f], and it is the enzymes of this venom which appear to cause severe destruction of tissue at the site of envenomation . In this manner enzymes appear to contribute to the toxicity of the whole venom. The local reactions of crude venoms make difficult their use as antigens in active immunization and in the production of antivenins . Data presented in this paper show that X-irradiated Ancistrodon piscivorus venom is less nectrotizing and less lethal than untreated venom, yet following irradiation this venom retains much of the antigenic character of the unirradiated venom. These observations suggest that irradiation of venoms may be a helpful step in the eventual production of an innocuous venom toxoid . SNAKES
MATERIALS and METHODS
The venom used was obtained from a colony of about 200 cottonmouths maintained at the United States Army Medical Research Laboratory . A glass receptacle in which the venom was collected was surrounded by crushed ice to facilitate rapid cooling of the venom (Fig. 1). Each week, venom was extracted from all snakes . After extraction and cooling the venom was lyophilized and stored at 4°C. A composite pool of lyophilized venom was then prepared from the quantity collected during a 4-month period . Venom used in this study was taken from this composite pool. Lethality of the crude venom was established by intravenous injection of approximately one hundred and fifty 18-20 gm female Swiss albino mice. The LD6o was found to be 335 mg per kg of mouse (Fig. 2). It has been demonstrated that dilute solutions of enzymes are inactivated by ionizing radiation more readily than those in the dry state or those in a concentrated solution [2j. 131
~ 32
HERSCHEL H. FLOWERS
A dilution of 1 mg venom, 10 ml distilled water was selected for exposure. After exposure, the irradiated venom solution was again lyophilized for testing purposes . For testing it was redissolved in saline in the desired concentration . Control samples not irradiated demonstrated a toxicity loss of less than 10 per cent upon being lyophilized again .
N
5
95
10
90
20
80
ó 30 á oc N
10 3 BO 50
50
30 80 90 ~ 3" 0
20 3"i
3ß
3"3
3"5 3"4 3"6 YENOM (mg/kg)
3 "7
3"B
~10
3"9
4" D
FIG. 2. LD fi p A . p(SClI'Olll.S VENOM 3'îS MG~KG
The X-ray machine employed was a North American Phillips 50 kV unit. No filter was used in order that maximum absorption of soft rays could be effected . The venom solution was exposed in 50 ml aliquots in a shallow glass dish. The depth of solution was maintained at 0~9 cm. A magnetic stirrer was employed to circulate the solution and provide
ao
0 0 S 30 x
O
1 2 3 4 6 EXPOSURE TIME FOR F~304 IN MINUTES
FIG. 3. X-IRRADIATION OF VENOM
6
FIG . ~ . COLLECTION OF VENOM .
~Ï0RIilAI YEN~II
,- ~
12Q~ S~ ~~
SAIINE COl~iROl
FIG . 4. COMPARATIVE HEMOLYSIS OF DOG RED BLOOD CELLS BY PHOSPHOLIPASE .
FIC . S. INTRADERMAL REACTIONS SEEN FROM THI ; SUBCUTANEOUS SURFACE OF RABBIT SKIN IN IECFED INTRADERMALLY WITH SERIAL DILUTIONS OF X-IRRADIATED VENOM (XV) IN COMPARISON WITH LIKE DILUTIONS OF' UNTREATED VENOM (NV) .
X-irradiation on Cottonmouth Moccasin
133
even exposure. The exposure time was 120 sec. Ferrous sulfate dosimetry was used in determining the total absorbed dose. For the 120 sec exposure the total absorbed dose was calculated to be 18,375 radf263 rad (Fig. 3). The procedure utilized for testing the irradiated and control venoms were 1 . A phospholipase activity test 2. Hemorrhagic activity test 3. Lethality tests 4. Immunoelectrophoresis and immunodiffusion The venoms of the snakes of the family Crotalidae yield a rich source of phospholipase (3] . In vivo this enzyme acts on tissue lecithin and produces lysolecithin . Lysolecithin is thought to be responsbile for the dissolution of red blood cells. To determine phospholi pase activity of the irradiated and control venom a procedure to measure in vitro hemolysis of RBC's was employed. The procedure was as follows : To supply lecithin for the production of lysolecithin an egg yolk-Tris buffer substrate was prepared . Fresh egg yolks were strained through gauze and added to an equal volume of Tris buffer (0~ 1 M pH 7~ 5 with CaCls added to 10-aM). This was thoroughly mixed, then centrifuged at 4,000 rpm for 1 to 1 1/2 hr. The supernatant was collected and bottled as substrate . Human or canine erythrocytes were washed three times in physiological saline, and a 50°.~° suspension was prepared for use in the test. Weighed quantities of venom were dissolved in Tris buffer containing CaCla. Successive dilutions were made with Tris buffer. A concentration of 1 mg venom per 100 ml buffer was adequate for venoms of high activity. Tubes were filled with 0~2 ml of substrate and 005 ml of venom. These were incubated for 45 min at 37°C. After incubating, 0~ 3 ml RBC's were added. After shaking gently to mix, it was incubated for 7 min at 37°C. On removal, 5 ml of cold saline was added to each tube. For accurate determinations it was important that each tube received the same incubation time, therefore, automatic pipetting equipment was employed to minimize the time required. Centrigufation in the cold for 10-20 min at 4,000 rpm packed the unhemolysed RBC's, and the supernatant was decanted. The supernatant was then diluted with a 1% Alconox* solution to reduce trubidity and optical density, convenient for reading on the lower half of the scale. The optical density was read at 540 um . Phospholipase activity was expressed as per cent of control value . The hemorrhagic activity was determined by utilizing the rabbit skin test as described by KONDO et al. [4] and OsAxr et al. [5] . This consisted of the normal and irradiated venom being injected intradermally into rabbits. After 24 hr the animal was sacrificed and the skin removed . The hemorrhagic areas on the underside of the skin were then compared . Differences were readily apparent and the results were photographically recorded. Lethality tests were accomplished by determining the LD6o I/V in 18-20 gm female Swiss albino mice. To demonstrate gross alteration of the antigenic character of the venom produced by X-irradiation, normal venom and X-irradiated venom were run against Polyvalent Antivenin (Crotalidae)t by agar immunodiffusion and by immunoelectrophoretic precipitation, The concentration of the venom samples was 10 mg/ml Tris buffer with CaClg. 'Alconox Detergent, Alconoz Inc., New York 3, N .Y. tPolyvalent Antivenin (Crotalidae), Wyeth Laboratories, Marietta, Pa.
l34
HERSCHEL H . FLOWERS
For immunoelectrophoresis, 1 by 3 in . miscroscope slides were covered with 2~5 to 3 ml of 1%, nutrient agar . After cooling a trough 1 ~5 mm wide and 254 cm long was cut in the agar. Three mm from the trough and on each side a circular well was cut . For immunoelectrophoresis the wells were filled with venom (005 ml instilled in each well) and the current applied for 2 hr at 150 V, 2 mA f0~6 mA ;cm gel), using barbital buffer (0~5 m pH 8~6) at 22 °C . After electrophoresis had separated the venom fractions, 0~ 15 ml of the polyvalent antivenin was instilled in the trough . Lines of precipitation appeared in the agar after 24 to 48 hr of incubation at 22°C . The slides were washed for 48 hr in NaCI to remove non-precipitated protein and then stained with amídoschwarz stain (1 °,~ in 7°.~ acetic acid) . The precipitation lines of the normal venom-antivenin reaction and the Xirradiated venom-antivenin reaction could then be compared and photographed . Immunodiffusion was accomplished in a similar manner, with the exception than an electrical current was not applied . The venom and antivenin were placed in individual wells in petri dishes filled with 15 ml agar and incubated at room temperature . Precipitation lines appeared after 18 to 24 hr, but precipitation was not complete until after 72 to 96 hr . At this time the petri dishes were photographed . Staining was unnecessary in this procedure because the lines of precipitation were very easily distinguished by eye . RESULTS Phospholipase was inactivated 95-100 per cent by radiation (Fig . 4) . The hemorrhagc skin test in rabbits demonstrated a decrease in the local effect of the X-irradiated venom as compared with normal venom (Fig. 5) . Both X-irradiated and normal venom in five dilutions from 1 :10 to I :160 in Tris bufferwereinjected intradermally . Only the higher concentrations of X-irradiated venom caused hemorrhage while all dilutions of normal venom caused hemorrhage. Immunodiffusion indicated some difference in the pattern of protein precipitation when tested against the polyvalent antivenin (Figs . 6 and 7) . Normal venom exhibited one large distinct band of precipitation with two to four smaller, less distinct bands behind the large one . The X-irradiated venom exhibited the same distinct large band, however, only one to three of the smaller bands were evident . These were less distinct than those demonstrated with the normal venom . Immunoelectrophoresis demonstrated much the same differences seen in the Immunodiffusion slides . The large band of precipitation was present in both the normal and Xirradiated venom . The X-irradiated venom exhibited fewer and more diffuse precipitation of the smaller bands . Perhaps the most significant alteration of venom affected by the X-irradiation was a decrease in lethality . After irradiation, 20 mg venom per kg mouse was required to produce the LDS° ; this is a sixfold decrease in lethality. This procedure was repeated numerous times with different samples of X-irradiated venom . Results ranged consistently from 198-205 mg/kg mouse . DISCUSSION Although studies have been accomplished concerning the effects of ionizing radiation on enzymes present in venom, to the best of the author's knowledge, only one instance is recorded in which whole venom was exposed to X-irradiatio~i . DUDLEY JACKSON [6J, in 1928, placed 45 mg of Crotalus atrox venom under l ;'2 in . of beef and exposed it to X-ray
FIG. Ó. COMPARATIVE PRECIPITATION BANDS FORMED IN AGAR BY X-IRRADIATED VENOM (XV) AND BY UNTREATED VENOM (NV) AGAINST POLYVALENT ANTIVENIN CROTALIDAE (PAC).
FIG . % . COMPARATIVE IMMUNOELECI'ROPHORETIC PATTERNS OF PRECIPITATION FORMED BY X IRRADIATED VENOM AND BY UNTREATED VENOM AGAINST CENTRALLY LOCATED POLYVALENT ANTIVENIN CROTALIDAE .
X-irradiation on Cottonmouth Moccasin
135
for 2 min. He then injected this material into two dogs. Both animals suffered severe local reactions and subsequent sloughing of tissue but did not die. There was no dosimetry reported with this exposure, nor can one be certain of the exact concentration of the venom injected; however, it is most likely that the concentration was much greater than the solutions used here. The present study demonstrates that the exposure of cottonmouth moccasin venom to X-irradiation decreases its local reactivity and lethal effect. These studies suggest that reduced local reactivity and lethal effect of this venom are accomplished with little change in the immunodiffusion and immunoelectrophoretic pattern of antigens. Although phospholipase was inactivated almost completely, the proteolytic enzymes remained capable of causing some hemorrhage and necrosis when injected intradermally, however, the activity of these enzymes was markedly reduced following irradiation. Thus, certain enzymes are totally inactivated while others appear to be only slightly effected . A need continues to exist for the development ofmethods useful in altering the lethality of venoms but which do not alter the antigenic character of the proteins. While several chemical agents have been employed to inhibit or minimize the local reactivity of the venom, none have been entirely successful in eliminating the lethal factors. Evidence has been presented which tends to support the belief that venoms contain, as yet unidentified, lethal factors which are not enzymic in character [7-9] . It appears possible to significantly inactivate this factor or factors by irradiation with little change in theimmunodiffusion and immuno-electrophoretic patterns of antigens. It seems improbable that the particular combination of irradiation dose and venom dilution used in this investigation represents conditions optimal for inactivation of toxicity with minimal alteration of antigenicity of the venom. It is indeed possible that other doses and dilutions may yield even greater inactivation with less effect on the antigenic character. For many years one of the greatest problems confronting researchers attempting to produce an effective antivenin to crotalid venoms has been that of the hemorrhage and local necrosis produced by the venom when injected into tissues. This has made difficult the production of antivenins of high titer and it has been an impediment to active immunization with crotalid venoms . It is, therefore, of importance that the use of ionizing radiation in the attenuation of venoms be further investigated. SUMMARY
1 . Exposure of a solution of 1 mg venom/10 ml distilled water for 120 sec (18,375 rad X263 rad) has yielded significant alterations in toxicity and enzyme activity. 2. The phospholipase has been inactivated 95-100 per cent . 3. Hemorrhagic and local nectrotizing action of the venom has been minimized. 4. It takes six times the weight of lyophilized X-irradiated venom to provide the mouse LDso as of normal venom. 5. Immunodiffusion and immunoelectrophoresis indicate little change in the precipitation pattern of antigens . Acknowledgements-The author is indebted to H. Bxews~e of the School of Medicine, University of Florida, for the development of the phospholipase determination procedure; and to Captain Goaoox MSC, Radiobiology Division, US Army Medical Research Laboratory, Fort Knox, Kentucky, for his technical assistance in the radiological aspects of this project. LODDE,
136
HERSCHEL H. FLOWERS
ZELLER, E. A.,
Advanc Etzymol. S,
REFERENCES 459, 1948 .
Parasitology, 36, 110, 1944 . Mvesncx, K., The Enzymes 4, pt . A. Academic
LEA, D. E., SMrrx, K. M., HOMES, B. and MAAKMAN, R., $LOTTA, K. H., BOYER, P. D., LARDY, H. and 1960.
[4] [5] [6] [~l [gl [9]
Koxno, H., Korroo, S., IKEZAWA, H. and MURATA, R.,
OFISAKA, H., IKEZAWA, H., KoNno, H., KONDO, S. and
Jap. J. Med. Sci. 13, 43, 1906. Ucxmn, N., Brit . J. erp. Path ., 41, 47,
W. T., J. Amer . vet., med. Ass., June 1928 . YANG, C. C., J. Formosan med. Ass., 59, 1326, 1960 . SLOTTA, K., FRANENKEL-CONRAT, H., Nature, Lond., 142, 213, 1938. GONCALVE3, J. M., L. G. Yieria Anal. Acad. Brasil. Cienc., 22, 140, 1950. JACKSON, D., HARRLSON,
Press.
1960 .