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African adders: partial characterization of snake venoms from three Bitis species of medical importance and their neutralization by experimental equine antivenoms.

Paixão-Cavalcante D, Kuniyoshi AK, Portaro FC, da Silva WD, Tambourgi DV - PLoS Negl Trop Dis (2015)

Bottom Line: The accidents are severe and the victims often have a poor prognosis due to the lack of effective specific therapies.Experimental antivenoms produced against B. arietans venom or Bitis g. rhinoceros plus B. nasicornis venoms cross-reacted with the venoms from the three species and blocked, in different degrees, all the enzymatic activities in which they were tested.We also demonstrated that horse antivenoms produced against B. arietans or B. g. rhinoceros plus B. nasicornis venoms can blocked some of the toxic activities of these venoms.

View Article: PubMed Central - PubMed

Affiliation: Immunochemistry Laboratory, Butantan Institute, São Paulo, São Paulo, Brazil.

ABSTRACT

Background: An alarming number of fatal accidents involving snakes are annually reported in Africa and most of the victims suffer from permanent local tissue damage and chronic disabilities. Envenomation by snakes belonging to the genus Bitis, Viperidae family, are common in Sub-Saharan Africa. The accidents are severe and the victims often have a poor prognosis due to the lack of effective specific therapies. In this study we have biochemically characterized venoms from three different species of Bitis, i.e., Bitis arietans, Bitis gabonica rhinoceros and Bitis nasicornis, involved in the majority of the human accidents in Africa, and analyzed the in vitro neutralizing ability of two experimental antivenoms.

Methodology/principal findings: The data indicate that all venoms presented phospholipase, hyaluronidase and fibrinogenolytic activities and cleaved efficiently the FRET substrate Abz-RPPGFSPFRQ-EDDnp and angiotensin I, generating angiotensin 1-7. Gelatinolytic activity was only observed in the venoms of B. arietans and B. nasicornis. The treatment of the venoms with protease inhibitors indicated that Bitis venoms possess metallo and serinoproteases enzymes, which may be involved in the different biological activities here evaluated. Experimental antivenoms produced against B. arietans venom or Bitis g. rhinoceros plus B. nasicornis venoms cross-reacted with the venoms from the three species and blocked, in different degrees, all the enzymatic activities in which they were tested.

Conclusion: These results suggest that the venoms of the three Bitis species, involved in accidents with humans in the Sub-Saharan Africa, contain a mixture of various enzymes that may act in the generation and development of some of the clinical manifestations of the envenomations. We also demonstrated that horse antivenoms produced against B. arietans or B. g. rhinoceros plus B. nasicornis venoms can blocked some of the toxic activities of these venoms.

No MeSH data available.


Related in: MedlinePlus

Enzymatic characterization of Bitis ssp venoms and the blockage of activities by antivenoms raised against B. arietans or B. g. rhinoceros plus B. nasicornis.[A]Hyaluronidase activity: B. arietans, B. g. rhinoceros and B. nasicornis venoms samples (30 μg) pre-incubated with hyaluronic acid at 37°C for 30 min. Alternatively, venom samples (30 μg) were pre-incubated with 10 μL of antivenoms raised against B. arietans venom (α-Ba) or B. g. rhinoceros plus B. nasicornis venoms (α-Br + Bn) for 30 min at RT, prior incubation with hyaluronic acid. The turbidity of the mixture was measured in a spectrophotometer at λem 405 nm. The results are representative of three individual experiments and expressed in units of turbidity reduction (UTR) per mg of venom. Statistical analyses were performed using two way Anova analysis (*P< 0.05). [B]Phospholipase A2activity: venom samples (2.5 μg) were added to the phospholipid FRET substrate and the fluorescence was immediately measured in a fluorimeter. Alternatively, venom samples (2.5 μg) were pre-incubated with 10 μL of antivenoms raised against B. arietans venom (α-Ba) or B. g. rhinoceros plus B. nasicornis venoms (α-Br + Bn) for 30 min at RT, prior incubation with phospholipid FRET substrate. The specific activity was expressed as UF/min/μg. Results are representative of 3 independent experiments performed in quadruplicate. Statistical analyses were performed using two way Anova (*P< 0.05).
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pntd.0003419.g002: Enzymatic characterization of Bitis ssp venoms and the blockage of activities by antivenoms raised against B. arietans or B. g. rhinoceros plus B. nasicornis.[A]Hyaluronidase activity: B. arietans, B. g. rhinoceros and B. nasicornis venoms samples (30 μg) pre-incubated with hyaluronic acid at 37°C for 30 min. Alternatively, venom samples (30 μg) were pre-incubated with 10 μL of antivenoms raised against B. arietans venom (α-Ba) or B. g. rhinoceros plus B. nasicornis venoms (α-Br + Bn) for 30 min at RT, prior incubation with hyaluronic acid. The turbidity of the mixture was measured in a spectrophotometer at λem 405 nm. The results are representative of three individual experiments and expressed in units of turbidity reduction (UTR) per mg of venom. Statistical analyses were performed using two way Anova analysis (*P< 0.05). [B]Phospholipase A2activity: venom samples (2.5 μg) were added to the phospholipid FRET substrate and the fluorescence was immediately measured in a fluorimeter. Alternatively, venom samples (2.5 μg) were pre-incubated with 10 μL of antivenoms raised against B. arietans venom (α-Ba) or B. g. rhinoceros plus B. nasicornis venoms (α-Br + Bn) for 30 min at RT, prior incubation with phospholipid FRET substrate. The specific activity was expressed as UF/min/μg. Results are representative of 3 independent experiments performed in quadruplicate. Statistical analyses were performed using two way Anova (*P< 0.05).

Mentions: Fig. 2A shows that the venoms from B. arietans, B. g. rhinoceros and B. nasicornis presented high, but not statistically different levels of hyaluronidase activity. The hyaluronidase activity determined for Bitis spp venoms was similar to the one detected in B. jararaca venom (12 UTR/mg). All Bitis venoms, tested in this study, presented similar phospholipase A2 activity. The activity of B. arietans was ~100 UF/min/μg, and of B. g. rhinoceros and B. nasicornis were ~80 UF/min/μg (Fig. 2B). The phospholipase activity of C. d terrificus venom, used as positive control of the assay, was 4–5 times higher than the Bitis venoms, i.e., 430 UF/min/μg.


African adders: partial characterization of snake venoms from three Bitis species of medical importance and their neutralization by experimental equine antivenoms.

Paixão-Cavalcante D, Kuniyoshi AK, Portaro FC, da Silva WD, Tambourgi DV - PLoS Negl Trop Dis (2015)

Enzymatic characterization of Bitis ssp venoms and the blockage of activities by antivenoms raised against B. arietans or B. g. rhinoceros plus B. nasicornis.[A]Hyaluronidase activity: B. arietans, B. g. rhinoceros and B. nasicornis venoms samples (30 μg) pre-incubated with hyaluronic acid at 37°C for 30 min. Alternatively, venom samples (30 μg) were pre-incubated with 10 μL of antivenoms raised against B. arietans venom (α-Ba) or B. g. rhinoceros plus B. nasicornis venoms (α-Br + Bn) for 30 min at RT, prior incubation with hyaluronic acid. The turbidity of the mixture was measured in a spectrophotometer at λem 405 nm. The results are representative of three individual experiments and expressed in units of turbidity reduction (UTR) per mg of venom. Statistical analyses were performed using two way Anova analysis (*P< 0.05). [B]Phospholipase A2activity: venom samples (2.5 μg) were added to the phospholipid FRET substrate and the fluorescence was immediately measured in a fluorimeter. Alternatively, venom samples (2.5 μg) were pre-incubated with 10 μL of antivenoms raised against B. arietans venom (α-Ba) or B. g. rhinoceros plus B. nasicornis venoms (α-Br + Bn) for 30 min at RT, prior incubation with phospholipid FRET substrate. The specific activity was expressed as UF/min/μg. Results are representative of 3 independent experiments performed in quadruplicate. Statistical analyses were performed using two way Anova (*P< 0.05).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4340965&req=5

pntd.0003419.g002: Enzymatic characterization of Bitis ssp venoms and the blockage of activities by antivenoms raised against B. arietans or B. g. rhinoceros plus B. nasicornis.[A]Hyaluronidase activity: B. arietans, B. g. rhinoceros and B. nasicornis venoms samples (30 μg) pre-incubated with hyaluronic acid at 37°C for 30 min. Alternatively, venom samples (30 μg) were pre-incubated with 10 μL of antivenoms raised against B. arietans venom (α-Ba) or B. g. rhinoceros plus B. nasicornis venoms (α-Br + Bn) for 30 min at RT, prior incubation with hyaluronic acid. The turbidity of the mixture was measured in a spectrophotometer at λem 405 nm. The results are representative of three individual experiments and expressed in units of turbidity reduction (UTR) per mg of venom. Statistical analyses were performed using two way Anova analysis (*P< 0.05). [B]Phospholipase A2activity: venom samples (2.5 μg) were added to the phospholipid FRET substrate and the fluorescence was immediately measured in a fluorimeter. Alternatively, venom samples (2.5 μg) were pre-incubated with 10 μL of antivenoms raised against B. arietans venom (α-Ba) or B. g. rhinoceros plus B. nasicornis venoms (α-Br + Bn) for 30 min at RT, prior incubation with phospholipid FRET substrate. The specific activity was expressed as UF/min/μg. Results are representative of 3 independent experiments performed in quadruplicate. Statistical analyses were performed using two way Anova (*P< 0.05).
Mentions: Fig. 2A shows that the venoms from B. arietans, B. g. rhinoceros and B. nasicornis presented high, but not statistically different levels of hyaluronidase activity. The hyaluronidase activity determined for Bitis spp venoms was similar to the one detected in B. jararaca venom (12 UTR/mg). All Bitis venoms, tested in this study, presented similar phospholipase A2 activity. The activity of B. arietans was ~100 UF/min/μg, and of B. g. rhinoceros and B. nasicornis were ~80 UF/min/μg (Fig. 2B). The phospholipase activity of C. d terrificus venom, used as positive control of the assay, was 4–5 times higher than the Bitis venoms, i.e., 430 UF/min/μg.

Bottom Line: The accidents are severe and the victims often have a poor prognosis due to the lack of effective specific therapies.Experimental antivenoms produced against B. arietans venom or Bitis g. rhinoceros plus B. nasicornis venoms cross-reacted with the venoms from the three species and blocked, in different degrees, all the enzymatic activities in which they were tested.We also demonstrated that horse antivenoms produced against B. arietans or B. g. rhinoceros plus B. nasicornis venoms can blocked some of the toxic activities of these venoms.

View Article: PubMed Central - PubMed

Affiliation: Immunochemistry Laboratory, Butantan Institute, São Paulo, São Paulo, Brazil.

ABSTRACT

Background: An alarming number of fatal accidents involving snakes are annually reported in Africa and most of the victims suffer from permanent local tissue damage and chronic disabilities. Envenomation by snakes belonging to the genus Bitis, Viperidae family, are common in Sub-Saharan Africa. The accidents are severe and the victims often have a poor prognosis due to the lack of effective specific therapies. In this study we have biochemically characterized venoms from three different species of Bitis, i.e., Bitis arietans, Bitis gabonica rhinoceros and Bitis nasicornis, involved in the majority of the human accidents in Africa, and analyzed the in vitro neutralizing ability of two experimental antivenoms.

Methodology/principal findings: The data indicate that all venoms presented phospholipase, hyaluronidase and fibrinogenolytic activities and cleaved efficiently the FRET substrate Abz-RPPGFSPFRQ-EDDnp and angiotensin I, generating angiotensin 1-7. Gelatinolytic activity was only observed in the venoms of B. arietans and B. nasicornis. The treatment of the venoms with protease inhibitors indicated that Bitis venoms possess metallo and serinoproteases enzymes, which may be involved in the different biological activities here evaluated. Experimental antivenoms produced against B. arietans venom or Bitis g. rhinoceros plus B. nasicornis venoms cross-reacted with the venoms from the three species and blocked, in different degrees, all the enzymatic activities in which they were tested.

Conclusion: These results suggest that the venoms of the three Bitis species, involved in accidents with humans in the Sub-Saharan Africa, contain a mixture of various enzymes that may act in the generation and development of some of the clinical manifestations of the envenomations. We also demonstrated that horse antivenoms produced against B. arietans or B. g. rhinoceros plus B. nasicornis venoms can blocked some of the toxic activities of these venoms.

No MeSH data available.


Related in: MedlinePlus