Toxicon, Vol. 33, No. IO. pp.

13874391, 1995
Copyright Q 1995 l&vin Scima Ltd
0041_010wmooo7&x
\ I
Printed in Great Britain. All rkhta rcservcd
co41-0101/95-59.50 + 0.00

IN r/lTRO ACTIVITY OF BaHl, THE MAIN HEMORRHAGIC

TOXIN OF BOTHROPS ASPER SNAKE VENOM ON BOVINE
ENDOTHELIAL CELLS

GAD1 BORKOW,’ JO& MARIA GUTIBRREZ2 and MICHAEL OVADIAl*

‘Department of Zoology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel;
and *Institute Clodomiro Picado, Facultad de Microbiologia Universidad de Costa Rica, Costa Rica

(Received 5 January 1995; accepted 12 May 1995)

G. Borkow, J. M. Gutierrez and M. Ovadia. In vitro activity of BaHl, the main

hemorrhagic toxin of Bothrops asper snake venom on bovine endothelial cells.
Toxicon 33, 1387-1391, 1995.-Incubation of BaHl, the main hemorrhagic
toxin purified from the venom of Bothrops asper, with endothelial cells caused
the appearance of spaces among the cells. This effect became more noticeable
with increasing hemorrhagin concentration and longer incubation time. Later,
the cells became rounded and detached from the substrate into the medium.
Augmentation of Trypan blue did not stain the detached cells, indicating their
viability. Moreover, after washing the floating cells from the toxin they could
be recultivated: they again spread on the substrate and proliferated, demon-
strating that BaHl is not directly cytotoxic to the endothelial cells.

The majority of snakebites in Central America are caused by pit vipers, among which
Bothrops asper is the most dangerous and abundant species (Minton, 1969; Bolanos, 1982).
It ranges from northern Mexico to the lowlands of Colombia and Ecuador (Martin, 1958;
Peters and Orejas-Miranda, 1970). The venom of this snake induces a complex series of
local effects including hemorrhage, myonecrosis and edema, in addition to systemic effects
such as hemorrhage, coagulation disorders, cardiovascular shock and acute renal failure
(Bolanos, 1982). Among the local and systemic effects caused by this venom, hemorrhage
is one of the most relevant as it develops extremely quickly after venom injection, causing
major microvasculature damage and blood loss which leads to muscle and other tissue
degeneration (Moreira et al., 1993). Similar striking consequences of envenomation, due
to the occurrence of local and peripheral hemorrhage, are caused by most crotalid and
viperid snake venoms (Ownby et al., 1978; Ohsaka, 1979).
In addition to four hemorrhagins previously isolated in this laboratory from the venoms
of Vipera palaestinae and Atractaspis engaddensis (Ovadia, 1978, 1987), we have recently
purified and characterized four hemorrhagic components from the venom of B. asper
(Borkow ef al., 1993; Gutierrez et al., 1995). BaHl, the main hemorrhagic metallo-
proteinase, is an acidic protein with pZ of 4.7 and a mol. wt of 64,000. The minimal

*Author to whom correspondence should be addressed.

1387
1388 Short Communications

hemorrhagic dose of BaH 1, which caused a 1 cm spot in ICR white mouse skin, is 0.18 pg,
with a specific activity 50 times higher than that of the whole venom (Borkow et al., 1993).
The literature on the hemorrhagic effects of either crude venom or purified hemorrhagic
toxins leaves one somewhat unclear as to whether there is only one or several mechanisms
by which these toxic metalloproteinases induce hemorrhage (Bjarnason and Fox, 1988-89).
Blood capillaries appear to be the main target of the hemorrhagic toxins, the result being
an alteration of the vessel permeability and extravasation. Ohsaka et al. (1973) and
Tsuchiya et al. (1974) attributed the hemorrhagic effect of the venom of Trimeresurus
jluvoviridis to enzymatic disruption of the basement membrane of the capillary wall,
followed by red blood cells oozing out through the interendothelial gaps. However, Ownby
et al. (1978) investigated the hemorrhagic toxins isolated from the venom of Crotulus utrox
and concluded that the primary effect of those hemorrhagic toxins was on the endothelial
cells themselves and the basement membrane around the capillaries was disrupted
afterwards. Erythrocytes escaped the capillary through the lysed endothelial cells and
basement membrane into the surrounding tissue. Similar observations were reported for
the hemorrhagic toxins isolated from venoms of Crotulus horridus (Ownby and Geren,
1987), Agkistrodon bilineatus (Ownby et al., 1990) and Cerustes cerustes (Rahmy et al.,
1992).
The controversy on the mechanism of action of snake metalloproteolytic hemorrhagins
may be due to the fact that previous studies were carried out in the complex system in vivo

Fig. 1. Effect of BaHl on endothelial cells.

(A) Control: endothelial cells without BaHl. (B) Endothelial cells after 2 hr of incubation with
BaHl (18 fig/ml). Spaces were formed between the cells. (C) Endothelial cells after 4 hr of
incubation with BaHl (18 pg/ml). The cells were detached from the substrate and became round.
(D) The detached round endothelial cells were centrifuged at 1000 rpm, washed twice with fresh
medium and recultivated in new wells. The cells spread normally, proliferated and formed a
monolayer.
 Short Communications 1389

and, therefore, suffered from at least two disadvantages: (1) the inability to distinguish
between the enzymatic and the cytotoxic activities of the hemorrhagic toxins because
in uiuo experiments do not allow follow-up of the sequence of events continuously under
the microscope; and (2) the inability to isolate the endothelial cells from all surrounding
tissues which may influence the hemorrhagic activity, although this influence may be
important. The significance of this work is, therefore, bound up with the focus on the
activity of BaHl on isolated endothelial cells cultured in vitro.
Primary bovine aortic endothelial cells were isolated as described by Schwartz (1978).
Briefly, segments of descending thoracic aorta, 20-30 cm in length, were rinsed in
phosphate-buffered saline (PBS), pH 7, containing 1% penicillin-streptomycin to remove
free blood. The aorta was incisioned lengthwise with sterile scissors until the inside lay flat
and it was then scraped gently with blade 21. The scraped endothelial cells were stirred
gently in D-Valine medium (Gibco) and cultured in flasks with this medium at 37°C in a
humidified iatmosphere of 95% air and 5% CO*. This medium inhibits the growth of
fibroblasts, thereby assuring an endothelial cell-enriched preparation. Every 3-4 days, the
cells were removed from the substrate using 0.25% trypsin containing 0.05% EDTA, and
recultivated in the same medium. After the 10th removal the medium used was changed
to DMEM, supplemented with 10% newborn calf serum, 1% L-glutamine and 1%
penicillin-streptomycin. For experimentation, the cells were grown in plates with 24 wells.
Each well contained about 10’ endothelial cells. The effect of BaHl on the cells was
monitored during the 48 hr following the addition of BaHl by two different methods: the
floating cells were counted by a cytometer; the remaining attached cells were also removed
by trypsinization and counted. The viability of both the attached and the floating cells was
examined after the addition of 30~1 of 0.05% Trypan blue in PBS.
BaHl (0.5-24.0 pg) was added to endothelial cells cultured in wells containing 0.5 ml
medium at 37°C and examined microscopically at various time intervals. The first visible
effect on the cell monolayer (Fig. 1A) was the appearance of spaces between the cells
(Fig. 1B) which became larger with longer incubation, until the cells detached from the
substrate into the medium and became rounded floating cells (Fig. 1C). This phenomenon
occurred more quickly as the toxin concentration used was increased (Fig. 2); 90-95% of

100

pglml BaHl
Fig. 2. Percentage of endothelial cells being detached from the bottom of the well after incubation
with various concentrations of BaH I (O-50 pg/ml).
The cells were incubated at 37°C and examined at various intervals (l-20 hr).
1390 Short Communications

the cells floated in the medium after 2 hr of incubation at concentrations of 48 pg/ml, but
this effect needed 4 hr at 12 pgg/ml or 20 hr at the lower concentration of 1 pg/ml. If the
floating cells were centrifuged at 100 x g and washed twice from the toxin with fresh
medium, the cells could be recultivated in new wells; they recuperated, spread normally
on the substrate and proliferated (Fig. lD), indicating that the hemorrhagic toxin was not
cytotoxic to the endothelial cells, but rather caused them to change their shape and to
separate from the substrate. These results concur with preliminary observations of this
group which indicated that the examined hemorrhagic toxin BaHl was not cytotoxic to
transformed murine endothelial cells, even at a toxin concentration as high as 65 pgg/ml
(Lomonte et al., 1994). Similar observations were found after incubation of various viperid
and crotalid crude venoms with isolated endothelial cells and on melanoma cells in vitro
(Borkow et al., 1994; Chaim-Matyas et al., 1987). In addition, previous ultrastructural
in vivo observations made by this group already indicated that the hemorrhage induced
by the whole venom of B. asper, or by its main hemorrhagin BaHl, included the
degradation of the basement membrane followed by the appearance of spaces between the
cells and by morphological alterations of the endothelial cells (Moreira et al., 1992, 1994).
The morphological changes and the detachment of the endothelial cells from the
substrate were inhibited by 20 mM EDTA and by 1 mM phenanthroline. Similarly, the
hemorrhagic activity of the whole venom of B. asper or its main hemorrhagic toxin BaHl
was inhibited by these metalloproteinase inhibitors (Borkow et al., 1994). However,
overnight incubation of the floating cells with 50 pgg/ml of BaHl resulted in the staining
of 90% of the cells by Trypan blue. Trypan blue did not stain the cells during the first
6 hr of incubation with BaHl even after they were detached from the substratum. As a
positive control for Trypan blue staining, the cells were incubated with cytotoxin P4
isolated from the venom of Naja nigricollis (Chaim-Matyas et al., 1991). This cytotoxin
P4 changed the permeability of the cell membrane within 1 hr, and allowed positive staining
of the attached cells by the Trypan blue.
The significance of this work is, therefore, linked with its contribution for the elucidation
of the mechanism of the hemorrhage induced by the crotalid venom. The early appearance
of spaces between the cultured endothelial cells (Fig. 1B) before any observable damage
to the cells further supports the hypothesis that the disruption of the basement membrane
is the primary effect, and may also explain the fast development of hemorrhage caused by
snake toxins. In vivo, the blood pressure probably accelerates the development of larger
spaces after the toxin begins the separation of the endothelial cells.
In conclusion, the present in vitro investigation shows that the significant hemorrhagic
toxin of B. asper (BaHl) is not directly cytotoxic to the bovine endothelial cells, but rather
causes the separation of the cells and their detachment from the surrounding substrate.

Acknowledgement-This study was supported by AID Grant No. DHR-5544-6-00-1064-00.

REFERENCES

Bjarnason, J. B. and Fox, J. W. (1988-89) Hemorrhagic toxins of snake venoms. J. Toxic.-Toxin Reo. 7, 121-209.
Bolanos, R. (1982) Las serpientes venonosas de Centroamerica y el problema de1 ofidismo. Primera part.
Aspectos zoologicos, epidemiologicos y biomedicos. Reota cost. Cienc. med. 3, 165-184.
Borkow, G., Gutierrez, J. M. and Ovadia, M. (1993) Isolation and characterization of synergistic hemorrhagins
from the venom of the snake Bofhrops asper. Toxicon 31, 1137-1150.
Borkow, G., Lomonte, B., Gutierrez, J. M. and Ovadia M. (1994) Effect of various viperidae and Crotalidae
snake venoms on endothelial cells in oitro. Toxicon 32, 1689-1695.
 Short Communications 1391

Chaim-Matyas, A. and Ovadia, M. (1987) Cytotoxic activity of various snake venoms on melanoma B16FlO and
chondrosarcoma. Life Sci. 40, 1601-1607.
Chaim-Matyas, A., Borkow, G. and Ovadia, M. (1991) Isolation and characterization of cytotoxin P4 from the
venom of Nqa nigricollis nigricollis preferentially active on tumor cells. Biochem. Int. 24, 415-421.
Gutitrrez, J. M., Diaz, C., Romero, M., Borkow, G. and Ovadia, M (1995) Isolation and characterization of
a metalloproteinase with a weak hemorrhagic activity from the venom of Bothrops asper. Toxicon 33, 19-29.
Lomonte, B., Gutitrrez, J. M., Borkow, G., Ovadia, M., Tarkowski, A. and Hanson, L. (1994) Activity of
hemorrhagic metalloproteinase BaHl and myotoxin II from Eothrops usper snake venom on capillary
endothelial cells in vitro. Toxicon 232, 505-510.
Martin, P. S. (1958) The biogeography of amphibians and reptiles in the Gomes Farias region. Tamaulipas,
Mexico. Univ. Michigan Misc. Publ. Mus. Zool.
Minton, S. A. (1969) Venomous Reptiles, p. 75. New York: Scribner’s Sons.
Moreira, L., Gutierrez., J. M., Borkow, G. and Ovadia, M. (1992) Ultrastructural alterations in mouSe capillary
blood vessels after experimental injections of venom from the snake Bothrops asper (terciopelo). Exp. Molec.
Path. 57, 124.-133.
Moreira, L., Borkow, G., Ovadia, M. and Gutitrrez, J. M. (1994) Pathological changes induced by BaHl, a
hemorrhagic proteinase isolated from Bothrops asper (terciopelo) snake venom, on mouse capillary blood
vessels. Toxicon 32, 977-987.
Ohsaka, A. (1979) Hemorrhagic, necrotizing and edema-forming effects of snake venoms. In: Snake Venoms,
pp. 480-546 (Lee, C. Y., Ed). Berlin: Springer.
Ohsaka, A., Just, M. and Habennann, E. (1973) Action of snake venom hemorrhagic principles on isolated
glomerular basement membrane. Biochim. biophys. Acra 323, 415-420.
Ovadia, M. (1978) Isolation and characterization of three hemorrhagic factors from the venom of Vipera
palaestinae. Toxicon 16, 479-487.
Ovadia, M. (1’987) Isolation and characterization of a hemorrhagic factor from the venom of the snake
Atractaspis engaddensis (Atractaspididae). Toxicon 25, 621-630.
Ownby, C. L. and Geren C. R. (1987) Pathogenesis of hemorrhage induced by hemorrhagic proteinase IV from
timber rattlesnake (Croralus atrox horridus) venom. Toxicon 25, 517-526.
Ownby, C. L., Bjamason, J. and Tu, A. T. (1978) Hemorrhagic toxins from rattlesnake (Crotalus atrox) venom.
Am. .I. Path. 93, 201-212.
Ownby, C. L., Nikai, T., Imai, K. and Sugihara, H. (1990) Pathogenesis of hemorrhage induced by bilotoxin,
a hemorrhagic toxin isolated from the venom of the common cantil (Aghistrodon bilineafus bihneatus). Toxicon
28, 837-846.
Peters, J. A. and Orejas-miranda, B. (1970) Catalogue of the Neotropical Squamata. Part 1: Snakes Bull. U.S.
Nat. Mus. 297, l-347.
Rahmy, T., Tu, A. T., El-Banhawey, M., El-Asmar, M. F. and Hassan, F. M. (1992) Cytopathologic effect of
Cerastes cerastes (Egyptian sand viper) venom and isolated hemorrhagic toxin on liver and kidney: an electron
microscopic Istudy. J. Nat. Toxins 1, 45-58.
Schwartz, S. M. (1978) Selection and characterization of bovine aortic endothelial cells. In Vitro 14, 966-970.
Tsuchiya, M., Ohshio, C., Ohashi, M., Ohsaka, A., Suzuki, K. and Fujishiro, Y. (1974) Cinematog,aphic and
electron microscopic analyses of the hemorrhage induced by the main hemorrhagic principle, HR-1, isolated
from the venom of Trimeresurusflavoviridis. In: Platelets, Thrombosis, and Inhibitors, Vol. 10, pp. 439-446
(Didisheim, I?. T., Shimamoto, T. and Yamazaki, H., Eds). Stuttgart: Schattauer.