Toxlcon Vol. 30, No . 3. pp . 3M-348, 1992. Printed in Great Britain .
0041-0101192 S5 .00 + .00 © 1992 Papmon Prey Pk
RHABDOMYONECROSIS EXPERIMENTALLY INDUCED IN WISTAR RATS BY AFRICANIZED BEE VENOM MARLSA M. AZEVEDO-MARQUES', DIRcEu B. FERREIRA2 and ROBERTO S. COSTA2* 'Departments of 'Internal Medicine, and Pathology, Faculty of Medicine, 14049 Ribeiráo Preto, SP, Brazil (Received 26 August
1991 ;
accepted 3 December
1991)
M. M. AzEvEDO-MARQuEs, D. B. FERREIRA and R. S. COSTA. Rhabdomyonecrosis experimentally induced in Wistar rats by Africanized bee venom. Toxicon 30, 344-348, 1992 .-Damage is reported to skeletal muscle experimentally induced in Wistar rats by Africanized bee venom (ABV) . Rhabdomyonecrosis was demonstrated indirectly by increased serum levels of the enzymes aspartate-aminotransferase and total creatine kinase, and directly by necrosis and inflammation observed by standard light microscopy of skeletal muscle . To our knowledge, this is the first report of a systemic damaging effect of ABV on skeletal muscle of experimentally envenomated rats. These data appear to reproduce experimentally some of the findings reported in cases of human envenomation due to multiple Africanized bee stings . AFRICAN bees were first introduced in Brazil in 1956 (GONCALVEs, 1974). After hybridizing with races existing in Brazil at the time, they spread as a crossbred race known as Africanized bees (ICERR et al., 1967 ; MIciHENER, 1975), crossed all of Central America and have recently reached North America (ELGART, 1990). Some reports of fatal cases of multiple Africanized bee stings have shown systemic alterations provoked by the venom (MERA et al., 1986; PATRICK et al., 1987). However, to our knowledge, there are no systematic studies, based on experimental models, related to envenomation by Africanized bees . In the present article, we describe changes in serum enzyme levels and pathological findings in skeletal muscle of Wistar rats inoculated i.m . or i.v. with Africanized bee venom (ABV). ABV was collected after sting extraction and venom sac expression (MELLO, 1970). The venom was then diluted 1 : 100 in 0.15 M saline at 4°C and utilized up to a maximum of 24 hr later. Each animal was injected with a dose of 1 .5 p1/100 g body wt (LDsp, 0.8 pl/100 g body wt by the i.v. route) . Sixty-four male albino rats of the Wistar strain weighing 90 to 100g were divided into two groups (experimental and control) of similar body wt and injected with ABV and saline, respectively . Intramuscular group: 24 animals injected i.m. (right thigh) with ABV and 24 injected with saline were sacrificed 4, 7, and 24 hr after inoculation . Intravenous group: eight animals injected i.v . with ABV into the lateral tail 0
Author to whom correspondence should be addressed .
Short Communications
FYo. 1 . Mtcxoscortc EnEc,-r or ABV (1 .5 p1/100g aonv wetoirr) RAIS.
345
oN sKmErAL
mLactB nv
WLSrAR
(A) Site of i .m. ÀBV injection into the right thigh muscle of an animal sacrificed 24 hr after inoculation showing extensive necrosis of skeletal muscle cells, oedema, and diffuse inflammatory infiltration with a predominance of mononuclear cells in the interstice . (B) Right thigh muscle from an anal that survived for 13 min after i .v. ABV injection showing partial loss of transversal strias and undulation and fragmentation of muscle fibres. (C) Same material as in (B) submitted to phosphotungstic haematoxylin staining. Note undulation and fragmentation of skeletal muscle fibres (arrows). Bais = 30 pm.
34 6
Short Communications \ 200or
ó W V W
i
1500 1000 500
Z W
AST -control /AST -utperknantol FIG.
2.
D CK - control M CK - fperirnaMol
ASPARTATE-AMINOTRANSFERASE (AST) AND TOTAL CREATING KINASE (CK) CONCENTRATIONS IN ABV-INJECTED ANIMALS AND CONTROLS .
was used at the dose of 1 .5 pl/100 g body weight . Concentrations are reported in units/litre for serum pools from the i .v . group (eight experimental animals) and from the i .m . groups (sacrificed 4, 7 and 24hr after ABV injection ; eight animals per time of sacrifice), and compared with serum pools from control animals (eight animals per group) . ABV
vein and sacrificed when they reached apneic condition over a period of time ranging from 70 sec to 13 min, and their respective controls injected with saline and pair sacrificed with the experimental animals. At the end of the observation period, the animals were sacrificed and 2 ml of blood was collected for serum enzyme measurement. Fragments of skeletal muscle removed from both thighs and from the ventral abdominal muscle were fixed by immersion in neutral 10% formalin solution and embedded in paraffin, and 6-,Um sections were cut, stained with haematoxylin and eosin (HE) and phosphotungstic haematoxylin (PTAH) and examined under the light microscope . The microscopic criteria used to characterize the lesion of the skeletal muscle fibres were those of HOMMA and Tu (1971) . Blood from each animal was allowed to clot for 2 hr at 4°C, centrifuged at 380g for 10 min and used for aspartate aminotransferase (AST) and total creatine kinase (CK) measurements . Equal volumes of serum from the eight animals in each group (injection route and time of sacrifice) were pooled and stored up to a maximum of 2 weeks. CK activity was measured by spectrophotometry using Granutest kit no . 12134 (MERCK S.A., Indiístrias Químicas, Brazil). The assay is based on phosphate transfer from phosphocreatine forming NADPH whose concentration is directly proportional to CK activity in serum. AST activity was measured by spectrophotometry using Merckotest kit no. 3362 (MERCK S.A., Indtístrias Quimicas, Brazil). The assay is based on amine group transfer from alpha-amino acids to alpha-ketoacids which is directly proportional to AST activity in serum. The results are reported as units/litre (UP) . All animals in the i.m . group survived until sacrifice. The microscopic examination of muscle at the site of inoculation showed extensive rhabdomyonecrosis associated with a diffuse acute inflammatory process and vascular congestion in all animals injected with ABV (Fig. IA). In contrast, microscopic examination of contralateral skeletal muscle and of the ventral abdominal wall of experimental animals and of all muscle specimens of control animals was normal . The serum enzyme levels measured in animals injected i.m .
Short Communications
347
are presented in Fig. 2. AST levels were 75 and 476, 72 and 487 and 68 and 391 for control and experimental animals, respectively, sacrificed 4, 7 and 24 hr after inoculation . CK levels were 423 and 1490, 149 and 220 and 126 and 149, respectively, for control and experimental animals sacrificed 4, 7 and 24 hr after inoculation. The eight animals injected i.v . with ABV survived 70 sec to 13 min after injection. Each of the animals in the control subgroup was pair sacrificed at the time of death of the respective experimental animal . Microscopic examination of skeletal muscle from animals injected i.v . showed rhabdomyonecrosis in all muscle specimens in four of the eight ABV-injected animals. These were the four animals with the longest survival (from 3.5 to 13 min) . Rhabdomyonecrosis showed a focal and segmental pattern involving some fibres and sparing others . The involved fibres showed partial fragmentation with loss of microfibrils and transversal striae . Intact muscle fibres alternated with elongated ones, presenting a homogeneous and hyperacidophilic cytoplasm and wavy and partially fragmented fibres . PTAH staining showed a loss of staining ability on the part of the involved fibres (Fig . 1 B, C) . Muscle fibre necrosis was not accompanied by an inflammatory infiltrate except in one animal, which was indeed the animal of longest survival (13 min) . The four remaining animals injected i.v . with ABV, who survived between 70 sec and 3 min, as well as the controls did not show microscopic changes in skeletal muscle . Serum enzyme levels measured in animals injected i.v. are presented in Fig. 2. AST levels were 85 and 440 and CK levels 386 and 1940 for control and experimental animals, respectively. Recently, ScxumAcHER et al (1990) demonstrated that the venoms of Africanized and European bees are similar in composition and effect . Indeed, the enzyme changes observed in the present study are comparable to those observed in cases of envenomation due to multiple bee stings (Humber et al., 1982 ; LIm et al., 1976). MEIIA et al . (1986) and PATRICK et al. (1987) reported victims of multiple Africanized bee stings who showed elevation of AST and CK levels and developed acute renal failure due to rhabdomyonecrosis and haemolysis . More recently, our group (AZEVEOO-MARQuEs et al., 1990; FERREIRA et al., 1990) reported four patients who died of multiple Africanized bee stings with extensive acute renal tubular necrosis and increased CK and AST levels. In the present study, damage to skeletal muscle was directly demonstrated by microscopic analysis of the muscle at the injection site (right thigh in the i.m . group) and of distant muscle (both thighs and abdominal wall in the i.v . group) . Microscopic examination of the injection site in the i.m . group revealed extensive rhabdomyonecrosis and an acute inflammatory reaction . A similar picture was reported in experimental envenomation with snake venom (HommA and Tu, 1971 ; GÜTIERREz and LomoNTE, 1989). The local necrotizing and inflammatory action of ABV is probably linked to melittin and phospholipase AZ. Hyaluronidase facilitates the diffusion of these substances, favouring their lyric action (HABERMANN, 1972 ; DOTIMAs and HIDER, 1987). In the present study we observed microscopic changes in the skeletal muscle of both thighs and of the ventral abdominal wall in four of the eight animals injected with ABV by the i.v. route, which survived longest. The absence of microscopic lesions of skeletal muscle in the four animals that survived less than 3 min demonstrates the need for a certain survival time for these changes to become established. These data demonstrate a systemic damaging effect of very early onset on skeletal muscle by i.v . injected ABV. To our knowledge, this is the first report of a damaging systemic effect on skeletal muscle by experimentally injected ABV. In contrast, animals injected i.m . did not present microscopic changes in skeletal muscle at a distance from the injection site, perhaps because of the dose used here and the possibility of inactivation of the venom by tissues at the site of
348
Short Communications
inoculation. These data appear to reproduce experimentally some of the findings reported in cases of human envenomation by multiple Africanized bee stings (MEDIA et al., 1986 ; AZEVEDO-MARQUES
et al ., 1990).
Acknowledgements-We are grateful to Mrs E. P. D. GUAL, M. HEREDIA, A. A. Funs and A. V. VERCESE for technical assistance. R. S. CosrA is a CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnol6gico) Fellow (grant no . 300462/91.3). This research was supported in part by FAPESP (Fundagio de Amparo i Pesquisa do Estado de Sio Paulo) .
REFERENCES AzEvEDo-MARQUM M. M., FERREIRA, D. B., CosTA, R. S., Gbpo, P. and HERING, S. E. (1990) Achados clinicos e laboratoriais de 4 acidentes fatais por m6ltiplas picadas de abelhas. Rev. Soc. Bras. Med. Trop. 23 (Suppl), 105-106. DOTmAs, E. M. and HIDER, R. C. (1987) Honeybee venom. Bee Wld 68, 51-70. EGART, G. W. (1990) Ant, bee and wasp stings . Dermatol. Clin . 8, 229-236. FERREIRA, D. B., SoAREs, F. A., CosTA, R. S., HERING, S. E., Cupo, P. and AZEVEDO-MARQUES, M. M. (1990) Míiltiplas picadas de abelhas: estudo anitomo-patol6gico de 4 acidentes fatais. Rev. Soc. Bras . Med. Trop. 23 (Suppl), 107 . GoNçALVps, L. S. (1974) The introduction of African Bees (Apis millijera adansonii) into Brazil and some comments on their spread in South America. Am . Bee J. 114, 414-415 . GuTmeREz, J. M. and LAmoNTE, B. (1989) Local damage induced by Bothrops snake venoms . A review. Mem. inst. Butantan 51, 211-223. HAIIERMANN, E. (1972) Bee and wasp venoms. The biochemistry and pharmacology of their peptides and enzymes are reviewed. Science 177, 314-322. HOmmA, M. and Tu, A. T. (1971) Morphology of local tissue damage in experimental snake envenomation . Br . J. exp. Path . 52, 538-542. HUmBLET, Y., SONNET, J. and VAN YPEQSE v DE STRWOU, C. (1982) Bee stings and acute tubular necrosis . Nephron 31, 187-188. KERB, W. E. (1967) The history of the introduction of African bees in Brazil. S. Am . Bee J. 39, 3-5. Lur, P., TAN, I. K. and FENG, P. H. (1976) Elevated serum enzymes in patients with wasp/bee sting and their clinical significance . Clin . Chirp . Acts 66, 405-409. MEtu, G., AQara .AEz , M., HENAo, J. E., SUS, A. A. and ARANoo, J. L. (1986) Acute renal failure due to multiple stings by Africanized bees . Ann. intern . Med. 104, 210-211. MS .m, M. L. S. (1970) A qualitative analysis of the protein in venoms from Apis meUijera (including A. m. adansonU) and Bombus atratus . J. Apic. Res. 9, 113-120. MrcuENEER, C. D. (1975) The Brazilian bee problem. A. Rev. Ent. 20, 399-416. PATRICK, A., RoBERTs, L., POON-KING, P. and JEELAL, V. (1987) Acute renal failure due to multiple stings by Africanized bees : report of the first case in Trinidad. W. l. med. J. 36, 43-44 . SCHUMACrmx, J. M., SCrMIDT, J. O., EDEN, N. B. and LowRY, J. E. (1990) Quantity, analysis and lethality of European and Africanized honey bee venoms. Am. J. trop . Med. hlyg. 43, 79-86.
0041-0101192 S5 .00 + .00 © 1992 Papmon Prey Pk
RHABDOMYONECROSIS EXPERIMENTALLY INDUCED IN WISTAR RATS BY AFRICANIZED BEE VENOM MARLSA M. AZEVEDO-MARQUES', DIRcEu B. FERREIRA2 and ROBERTO S. COSTA2* 'Departments of 'Internal Medicine, and Pathology, Faculty of Medicine, 14049 Ribeiráo Preto, SP, Brazil (Received 26 August
1991 ;
accepted 3 December
1991)
M. M. AzEvEDO-MARQuEs, D. B. FERREIRA and R. S. COSTA. Rhabdomyonecrosis experimentally induced in Wistar rats by Africanized bee venom. Toxicon 30, 344-348, 1992 .-Damage is reported to skeletal muscle experimentally induced in Wistar rats by Africanized bee venom (ABV) . Rhabdomyonecrosis was demonstrated indirectly by increased serum levels of the enzymes aspartate-aminotransferase and total creatine kinase, and directly by necrosis and inflammation observed by standard light microscopy of skeletal muscle . To our knowledge, this is the first report of a systemic damaging effect of ABV on skeletal muscle of experimentally envenomated rats. These data appear to reproduce experimentally some of the findings reported in cases of human envenomation due to multiple Africanized bee stings . AFRICAN bees were first introduced in Brazil in 1956 (GONCALVEs, 1974). After hybridizing with races existing in Brazil at the time, they spread as a crossbred race known as Africanized bees (ICERR et al., 1967 ; MIciHENER, 1975), crossed all of Central America and have recently reached North America (ELGART, 1990). Some reports of fatal cases of multiple Africanized bee stings have shown systemic alterations provoked by the venom (MERA et al., 1986; PATRICK et al., 1987). However, to our knowledge, there are no systematic studies, based on experimental models, related to envenomation by Africanized bees . In the present article, we describe changes in serum enzyme levels and pathological findings in skeletal muscle of Wistar rats inoculated i.m . or i.v. with Africanized bee venom (ABV). ABV was collected after sting extraction and venom sac expression (MELLO, 1970). The venom was then diluted 1 : 100 in 0.15 M saline at 4°C and utilized up to a maximum of 24 hr later. Each animal was injected with a dose of 1 .5 p1/100 g body wt (LDsp, 0.8 pl/100 g body wt by the i.v. route) . Sixty-four male albino rats of the Wistar strain weighing 90 to 100g were divided into two groups (experimental and control) of similar body wt and injected with ABV and saline, respectively . Intramuscular group: 24 animals injected i.m. (right thigh) with ABV and 24 injected with saline were sacrificed 4, 7, and 24 hr after inoculation . Intravenous group: eight animals injected i.v . with ABV into the lateral tail 0
Author to whom correspondence should be addressed .
Short Communications
FYo. 1 . Mtcxoscortc EnEc,-r or ABV (1 .5 p1/100g aonv wetoirr) RAIS.
345
oN sKmErAL
mLactB nv
WLSrAR
(A) Site of i .m. ÀBV injection into the right thigh muscle of an animal sacrificed 24 hr after inoculation showing extensive necrosis of skeletal muscle cells, oedema, and diffuse inflammatory infiltration with a predominance of mononuclear cells in the interstice . (B) Right thigh muscle from an anal that survived for 13 min after i .v. ABV injection showing partial loss of transversal strias and undulation and fragmentation of muscle fibres. (C) Same material as in (B) submitted to phosphotungstic haematoxylin staining. Note undulation and fragmentation of skeletal muscle fibres (arrows). Bais = 30 pm.
34 6
Short Communications \ 200or
ó W V W
i
1500 1000 500
Z W
AST -control /AST -utperknantol FIG.
2.
D CK - control M CK - fperirnaMol
ASPARTATE-AMINOTRANSFERASE (AST) AND TOTAL CREATING KINASE (CK) CONCENTRATIONS IN ABV-INJECTED ANIMALS AND CONTROLS .
was used at the dose of 1 .5 pl/100 g body weight . Concentrations are reported in units/litre for serum pools from the i .v . group (eight experimental animals) and from the i .m . groups (sacrificed 4, 7 and 24hr after ABV injection ; eight animals per time of sacrifice), and compared with serum pools from control animals (eight animals per group) . ABV
vein and sacrificed when they reached apneic condition over a period of time ranging from 70 sec to 13 min, and their respective controls injected with saline and pair sacrificed with the experimental animals. At the end of the observation period, the animals were sacrificed and 2 ml of blood was collected for serum enzyme measurement. Fragments of skeletal muscle removed from both thighs and from the ventral abdominal muscle were fixed by immersion in neutral 10% formalin solution and embedded in paraffin, and 6-,Um sections were cut, stained with haematoxylin and eosin (HE) and phosphotungstic haematoxylin (PTAH) and examined under the light microscope . The microscopic criteria used to characterize the lesion of the skeletal muscle fibres were those of HOMMA and Tu (1971) . Blood from each animal was allowed to clot for 2 hr at 4°C, centrifuged at 380g for 10 min and used for aspartate aminotransferase (AST) and total creatine kinase (CK) measurements . Equal volumes of serum from the eight animals in each group (injection route and time of sacrifice) were pooled and stored up to a maximum of 2 weeks. CK activity was measured by spectrophotometry using Granutest kit no . 12134 (MERCK S.A., Indiístrias Químicas, Brazil). The assay is based on phosphate transfer from phosphocreatine forming NADPH whose concentration is directly proportional to CK activity in serum. AST activity was measured by spectrophotometry using Merckotest kit no. 3362 (MERCK S.A., Indtístrias Quimicas, Brazil). The assay is based on amine group transfer from alpha-amino acids to alpha-ketoacids which is directly proportional to AST activity in serum. The results are reported as units/litre (UP) . All animals in the i.m . group survived until sacrifice. The microscopic examination of muscle at the site of inoculation showed extensive rhabdomyonecrosis associated with a diffuse acute inflammatory process and vascular congestion in all animals injected with ABV (Fig. IA). In contrast, microscopic examination of contralateral skeletal muscle and of the ventral abdominal wall of experimental animals and of all muscle specimens of control animals was normal . The serum enzyme levels measured in animals injected i.m .
Short Communications
347
are presented in Fig. 2. AST levels were 75 and 476, 72 and 487 and 68 and 391 for control and experimental animals, respectively, sacrificed 4, 7 and 24 hr after inoculation . CK levels were 423 and 1490, 149 and 220 and 126 and 149, respectively, for control and experimental animals sacrificed 4, 7 and 24 hr after inoculation. The eight animals injected i.v . with ABV survived 70 sec to 13 min after injection. Each of the animals in the control subgroup was pair sacrificed at the time of death of the respective experimental animal . Microscopic examination of skeletal muscle from animals injected i.v . showed rhabdomyonecrosis in all muscle specimens in four of the eight ABV-injected animals. These were the four animals with the longest survival (from 3.5 to 13 min) . Rhabdomyonecrosis showed a focal and segmental pattern involving some fibres and sparing others . The involved fibres showed partial fragmentation with loss of microfibrils and transversal striae . Intact muscle fibres alternated with elongated ones, presenting a homogeneous and hyperacidophilic cytoplasm and wavy and partially fragmented fibres . PTAH staining showed a loss of staining ability on the part of the involved fibres (Fig . 1 B, C) . Muscle fibre necrosis was not accompanied by an inflammatory infiltrate except in one animal, which was indeed the animal of longest survival (13 min) . The four remaining animals injected i.v . with ABV, who survived between 70 sec and 3 min, as well as the controls did not show microscopic changes in skeletal muscle . Serum enzyme levels measured in animals injected i.v. are presented in Fig. 2. AST levels were 85 and 440 and CK levels 386 and 1940 for control and experimental animals, respectively. Recently, ScxumAcHER et al (1990) demonstrated that the venoms of Africanized and European bees are similar in composition and effect . Indeed, the enzyme changes observed in the present study are comparable to those observed in cases of envenomation due to multiple bee stings (Humber et al., 1982 ; LIm et al., 1976). MEIIA et al . (1986) and PATRICK et al. (1987) reported victims of multiple Africanized bee stings who showed elevation of AST and CK levels and developed acute renal failure due to rhabdomyonecrosis and haemolysis . More recently, our group (AZEVEOO-MARQuEs et al., 1990; FERREIRA et al., 1990) reported four patients who died of multiple Africanized bee stings with extensive acute renal tubular necrosis and increased CK and AST levels. In the present study, damage to skeletal muscle was directly demonstrated by microscopic analysis of the muscle at the injection site (right thigh in the i.m . group) and of distant muscle (both thighs and abdominal wall in the i.v . group) . Microscopic examination of the injection site in the i.m . group revealed extensive rhabdomyonecrosis and an acute inflammatory reaction . A similar picture was reported in experimental envenomation with snake venom (HommA and Tu, 1971 ; GÜTIERREz and LomoNTE, 1989). The local necrotizing and inflammatory action of ABV is probably linked to melittin and phospholipase AZ. Hyaluronidase facilitates the diffusion of these substances, favouring their lyric action (HABERMANN, 1972 ; DOTIMAs and HIDER, 1987). In the present study we observed microscopic changes in the skeletal muscle of both thighs and of the ventral abdominal wall in four of the eight animals injected with ABV by the i.v. route, which survived longest. The absence of microscopic lesions of skeletal muscle in the four animals that survived less than 3 min demonstrates the need for a certain survival time for these changes to become established. These data demonstrate a systemic damaging effect of very early onset on skeletal muscle by i.v . injected ABV. To our knowledge, this is the first report of a damaging systemic effect on skeletal muscle by experimentally injected ABV. In contrast, animals injected i.m . did not present microscopic changes in skeletal muscle at a distance from the injection site, perhaps because of the dose used here and the possibility of inactivation of the venom by tissues at the site of
348
Short Communications
inoculation. These data appear to reproduce experimentally some of the findings reported in cases of human envenomation by multiple Africanized bee stings (MEDIA et al., 1986 ; AZEVEDO-MARQUES
et al ., 1990).
Acknowledgements-We are grateful to Mrs E. P. D. GUAL, M. HEREDIA, A. A. Funs and A. V. VERCESE for technical assistance. R. S. CosrA is a CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnol6gico) Fellow (grant no . 300462/91.3). This research was supported in part by FAPESP (Fundagio de Amparo i Pesquisa do Estado de Sio Paulo) .
REFERENCES AzEvEDo-MARQUM M. M., FERREIRA, D. B., CosTA, R. S., Gbpo, P. and HERING, S. E. (1990) Achados clinicos e laboratoriais de 4 acidentes fatais por m6ltiplas picadas de abelhas. Rev. Soc. Bras. Med. Trop. 23 (Suppl), 105-106. DOTmAs, E. M. and HIDER, R. C. (1987) Honeybee venom. Bee Wld 68, 51-70. EGART, G. W. (1990) Ant, bee and wasp stings . Dermatol. Clin . 8, 229-236. FERREIRA, D. B., SoAREs, F. A., CosTA, R. S., HERING, S. E., Cupo, P. and AZEVEDO-MARQUES, M. M. (1990) Míiltiplas picadas de abelhas: estudo anitomo-patol6gico de 4 acidentes fatais. Rev. Soc. Bras . Med. Trop. 23 (Suppl), 107 . GoNçALVps, L. S. (1974) The introduction of African Bees (Apis millijera adansonii) into Brazil and some comments on their spread in South America. Am . Bee J. 114, 414-415 . GuTmeREz, J. M. and LAmoNTE, B. (1989) Local damage induced by Bothrops snake venoms . A review. Mem. inst. Butantan 51, 211-223. HAIIERMANN, E. (1972) Bee and wasp venoms. The biochemistry and pharmacology of their peptides and enzymes are reviewed. Science 177, 314-322. HOmmA, M. and Tu, A. T. (1971) Morphology of local tissue damage in experimental snake envenomation . Br . J. exp. Path . 52, 538-542. HUmBLET, Y., SONNET, J. and VAN YPEQSE v DE STRWOU, C. (1982) Bee stings and acute tubular necrosis . Nephron 31, 187-188. KERB, W. E. (1967) The history of the introduction of African bees in Brazil. S. Am . Bee J. 39, 3-5. Lur, P., TAN, I. K. and FENG, P. H. (1976) Elevated serum enzymes in patients with wasp/bee sting and their clinical significance . Clin . Chirp . Acts 66, 405-409. MEtu, G., AQara .AEz , M., HENAo, J. E., SUS, A. A. and ARANoo, J. L. (1986) Acute renal failure due to multiple stings by Africanized bees . Ann. intern . Med. 104, 210-211. MS .m, M. L. S. (1970) A qualitative analysis of the protein in venoms from Apis meUijera (including A. m. adansonU) and Bombus atratus . J. Apic. Res. 9, 113-120. MrcuENEER, C. D. (1975) The Brazilian bee problem. A. Rev. Ent. 20, 399-416. PATRICK, A., RoBERTs, L., POON-KING, P. and JEELAL, V. (1987) Acute renal failure due to multiple stings by Africanized bees : report of the first case in Trinidad. W. l. med. J. 36, 43-44 . SCHUMACrmx, J. M., SCrMIDT, J. O., EDEN, N. B. and LowRY, J. E. (1990) Quantity, analysis and lethality of European and Africanized honey bee venoms. Am. J. trop . Med. hlyg. 43, 79-86.
