CYTOTOXICITY AND MORPHOLOGICAL ANALYSIS OF CELL DEATH INDUCED BY Bothrops VENOMS FROM THE NORTHEAST OF ARGENTINA

Bothrops snake venoms have been proved toxic to a variety of cell types, in both in vivo and in vitro models. Studies on the pharmacological actions of Bothrops venoms from Argentina are relatively scarce and the direct action of the crude venoms has not been assessed using cell culture models. In this work, we investigated the cytotoxicity of crude venoms from B. alternatus and B. diporus in a skeletal muscle (C2C12) cell line, which is commonly used as a model for studying the myotoxic action of snake venom. Both venoms (1.25-50 μg/mL) induced an early and significant decrease in cell viability. The cytotoxic concentration 50 (CC ), determined three hours after exposure, revealed that B. diporus venom was significantly more cytotoxic (CC : 2 μg/mL) than B. alternatus (CC : 5.8 μg/mL). To investigate the cell death mechanism involved, myoblast cells were examined by phase contrast microscopy and after acridine orange and ethidium bromide fluorescence staining, respectively. Our data clearly demonstrated that an apoptotic mechanism mediated this cell line destruction. The current study aimed to provide new information on the cytotoxicity mechanisms of Argentine Bothrops snake venoms on a skeletal muscle cell line. 50


INTRODUCTION
In snakebites cases, envenomations are associated with an amazing variety of pathophysiological manifestations, which differ among the various groups of snakes and even within the same genera and species (1).Acute skeletal muscle damage is a frequent manifestation in envenomations induced by Elapidae and Viperidae snake families (1,2).
The majority of snakebites in northeastern Argentina are caused by B. alternatus (yarará grande) and B. diporus (yarará chica), reptiles that belong to the Viperidae family (3)(4)(5).Their venoms induce predominantly local myotoxicity, characterized by myonecrosis often associated with other effects, such as hemorrhage, blistering and edema, in a complex pattern of local tissue damage (6,7).Myonecrosis is an important medical complication of snakebites that can lead to drastic sequelae including permanent tissue loss, disability, amputation or myoglobinuria and acute renal failure, a frequent cause of death in snakebite victims (8).
Previous works from our group demonstrated in vivo myotoxic effects of Bothrops alternatus venom in a rat gastrocnemius muscle.Muscular necrosis, hemorrhage among muscular fibers and edema were described (9,10).
Although in vivo experiments are adequate techniques to investigate the action of snake venoms on the overall muscle tissue scenario, these models preclude the analysis of some specific aspects of the toxin action on muscle cells.In that sense, as an approach to the characterization of the mechanisms involved in myotoxicity, cultured cells (11,12) or isolated enzymes -such as myotoxic PLA 2 s - (13)(14)(15)(16)(17) have been evaluated as targets for crude venoms.
On the other hand, in vitro cell culture model and screening assays for cell death have become important tools to determine the chronology of events resulting in apoptosis (18).
Despite the great advances in the understanding of morphological and biochemical alterations associated with chemically induced cell injury, the in vitro cytotoxic effects of Botrhops venoms from Argentina, at the cellular level, have not been characterized yet.
C2C12 is a cell line that exhibits most of the characteristics of normal myoblastic cells (19).These cells are commonly used as a model for studying muscle cell growth and differentiation.On the other hand, they are also a suitable target for the cytotoxic action of crude venoms or isolated enzymes, as proven by previous studies (11,12,17,(20)(21)(22)(23).Therefore, this cell culture is an appropriate in vitro model system to evaluate the myotoxicity and the events involved in the cell death caused by Bothrops venoms.
In the current work we compared the toxic potency between both venoms on myoblast cell line and, in order to elucidate a putative direct cytotoxic action, we analysed the cytotoxicity and morphological features of cell death induced by B. alternatus and B. diporus venoms on the mouse myoblast continuous cell line, C2C12.

Venoms
B. alternatus and B. diporus crude venoms were collected from several snakes from northeastern Argentina, vacuum dried and kept at -20°C.

Cell Culture
C2C12 (CRL-1772, ATCC) is a subclone murine skeletal muscle cell line derived from mouse myoblast cells obtained from normal adult C3H leg muscle.This cell line rapidly differentiates and produces extensive contracting myotubes that express characteristic muscle proteins.Cytolysis was assessed in undifferentiated myoblasts.

Cytotoxicity Assays: CC 50 Determination
For cytotoxicity assays, undifferentiated myoblasts were harvested from subconfluent monolayers after exposure to trypsin/EDTA at 37°C.The resuspended cells were seeded in 96-well microplates, at an approximate initial density of 1 to 2 x 10 4 cells per well, on the same growth medium.When monolayers reached 80% subconfluence, different concentrations of Bothrops venoms, diluted in sterile culture medium supplemented with 5% of FBS (ranging from 1.25 to 50 µg/mL), were added to the cell cultures (after aspirating their medium) in a total volume of 200 µL/well.
After three hours of incubation, cell viability was quantified by crystal violet staining, non-adherent cells were removed by washing twice with phosphate-buffered saline (PBS) and adherent cells were fixed with methanol:glacial acetic acid (3:1 ratio), and stained with 0.5% crystal violet in 20% (v/v) methanol.The dye was released from the cells by addition of ethanol: glacial acetic acid (3:1 ratio).The optical density of the released dye solution was determined at 620 nm.Cytotoxicity was determined by comparing the resulting absorbances with the mean absorbance of the control wells (without venom, considered as 100% viability), and was expressed as percentage of cell viability.The 50% cytotoxic concentration (CC 50 ) is defined as the quantity of venom generating 50% of cell viability, compared to the control.The values of the percentages of cell viability were plotted against venom concentrations, and CC 50 was determined.Experiments were carried out in quadruplicate.

Cell Morphology Analysis
Morphological alterations and cell damage were qualitatively investigated using a light phase contrast microscope (Axiovert 40®, Carl Zeiss Argentina), and the photos were taken with a digital camera (Canon CCD 2272x1704, Argentina).

Apoptosis Assay
In order to determine whether B. alternatus and B. diporus venoms induce apoptosis in this myoblast cell line, cells were grown on coverslides and treated with 10 µg/mL of Bothrops venoms for 30 minutes at 37°C and 5% CO 2 .This concentration, higher than the CC 50 obtained after the three-hour incubation, was chosen bearing in mind the short exposure time in this apoptosis assay.After incubation, cultured myoblast were washed twice with PBS and gently mixed with a mixture of acridine orange (AO) (1 µg/mL) and ethidium bromide (EB) (1 µg/mL) dye solution for one minute as described by Spector et al. (24).Coverslides were applied to the slides; afterwards, the sections were observed and photographed under a fluorescence microscope (Axioskop 40®/Axioskop 40 FL®, Carl Zeiss, Argentina).

Statistical Analysis
Data represent the mean ± standard deviation (SD) of at least four replications.
Statistical significance was tested by one-way ANOVA and Tukey (HSD) and pvalues inferior to 0.05 were considered significant.

Cytotoxicity
The toxicity of Bothrops venoms was assessed quantitatively by a crystal violet dye uptake assay.After three hours of treatment, a dose-dependent cell death was induced by both venoms, being cell viability significantly inhibited, regarding the untreated control, at all venom concentrations assayed (Figure 1 -A

Cell Morphology
To assess alterations of cell morphology subsequent to treatments, C2C12 cells were grown on glass coverslides.Untreated C2C12 cells were homogeneously distributed on cultured field; they exhibited a thin and elongated shape, observed by phase-contrast microscopy (Figure 2 -A, B and C).However, after incubation with10 µg/mL of B. alternatus venom -a concentration that produced detectable changesthe occurrence of various morphological abnormalities was recorded.The 30-minute alterations, induced by the treatment, included reduction of nuclear sizes, which often displayed diverse degrees of chromatin condensation.Cell rounding and some areas devoid of cells were also noticed in the same culture (Figure 2 -D).Furthermore, cell shrinkage and the formation of blebs on cell surface finally resulted in the generation of apoptotic bodies (Figure 2 -E and F).Similar observations were obtained with B. diporus venom treatment (data not shown).

Apoptosis Assay
In order to determined whether B. alternatus and B. diporus venoms induced morphological alterations that could be attributed to an apoptotic mechanism, myoblast cells were treated with the venoms for short periods and then stained with the nucleic acid-binding fluorochromes, acridine orange and ethidium bromide.

DISCUSSION
In vivo experiments are adequate models to investigate the action of snake venoms on muscle tissue.However, these models preclude the analysis of specific aspects related to the action of myotoxins on muscle cells.Cell culture models have been northeast of Argentina.J Venom Anim Toxins incl Trop Dis.2009;15(1):37 utilized aiming at the dissection of discrete steps in muscle cell damage; keeping in mind that extrapolation to in vivo conditions must be done with caution (25).Studies employing a variety of cell lines in culture showed that snake venoms display a relatively broad cytotoxic spectrum (11,12,26,27).Similar observations were made with several toxins, mostly myotoxic PLA 2 s isolated from viperid snake venoms (15,17,21,23,(28)(29)(30).
Thus, due to the possibility of manipulating cellular processes and mechanisms in vitro, myoblast cell culture allows the study of specific aspects of venoms action on these cells.
In this study, we demonstrated a direct cytotoxic effect of B. alternatus and B. diporus snake crude venoms on a skeletal muscle myoblast cell line.The decrease in cell viability, observed following exposure to Bothrops venoms, suggest a cytotoxic activity of both venoms in vitro.Our observations fully agree with experimental evidence of rapid and drastic degenerative events that occur in skeletal muscle fibers in vivo (10).Interestingly, our data are very similar to those reported by Oliveira et al.Several works demonstrated that crude elapid and crotalic venoms induce apoptosis in different cell types (11,31,32) or isolated enzymes from bothropic venoms, such as phospholipases A 2 (33,34) and metalloproteases (35)(36)(37)(38).Cell death is currently the subject of a considerable number of investigations.This interest stems, in part, from the potential of understanding oncogenesis and from the possibility of exploiting cell death program for therapeutic purposes (39)(40)(41).
In this work, we studied the in vitro process of cell death caused by B. alternatus and B. diporus venoms on C2C12 cell line, observing that a very short exposure of cells to these venoms results in marked morphological changes consistent with an apoptotic mechanism of cell death, such as chromatin condensation and nuclear fragmentation, cell shrinkage and bleb formation on cell surface, which finally results in the generation of apoptotic bodies.The role of apoptosis in tissue-damaging effects induced by these venoms requires further studies.
In conclusion, this is a first-hand investigation showing the potential cytotoxic action and the apoptotic effect of Bothrops venoms in the C2C12 cell line.

Figure 1 .
Figure 1.Decrease in cell viability of C2C12 cells three hours after the inoculation with Bothrops crude venoms (1.25 to 50 µg/mL); exposure assessed by a crystal violet cell uptake assay (A).The mean of the control group, treated with medium alone, was taken as 100% of dye uptake value.Data (mean + SD) are representative of three independent experiments carried in quadruplicate.Determination of cytotoxic concentration 50 (CC 50 ) (B). * p < 0.05: B. alternatus and B. diporus versus control group; † p < 0.05: B. alternatus versus B. diporus venom (one-way Anova and Tukey).

Figure 2 .
Figure 2. Morphological characterization under phase contrast microscopy of cell death induced by B. alternatus venom in murine C2C12 cells.Control cells (respectively 200, 400 and 1,000x) (A, B and C).Irregularity in shape and cellular detachment in treated cells (200x) (D).Cell shrinkage and formation of blebs on the cell surface (400x) (E).Apoptotic body (arrow, 1,000x) (F).
Control untreated cells exhibited a green fluorescence, due to exclusion of ethidium bromide but not of acridine orange.Viable cells showed a light green nucleus with intact structure and presented punctuate orange red fluorescence in the cytoplasm, representing lysosomes stained by acridine orange (Figure3, control).After 30 minutes of incubation with 10 µg/mL of Bothrops venoms, typical features of apoptosis were observed.Apoptotic cells exhibited a bright green nucleus (showing condensation of chromatin), dense green areas and evident membrane blebbing (Figure 3, B. alternatus and B. diporus).Some condensed nuclei were stained with orange fluorescent dye with ethidium bromide, indicating compromised membrane integrity, probably produced after the first stages of nuclear apoptotic condensation, mainly in the case of B. diporus venom (Figure 3, B. diporus).

Figure 3 .
Figure 3. Representative photomicrographs showing cell shrinkage, chromatin condensation and the formation of blebs on the cell surface associated with apoptosis assayed by acridine orange and ethidium bromide staining.Cell lines were grown on coverslides and treated for 30 minutes with 10 µg/mL of Bothrops venoms.