http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, 2015; 53(6): 930–934 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.950385
SHORT COMMUNICATION
In vitro and in vivo genotoxic evaluation of Bothrops moojeni snake venom Nathalia Novak Zobiole1, Thiago Caon2, Je´ssica Wildgrube Bertol2, Cintia Alves de Souza Pereira1, Brunna Mary Okubo1, Susana Elisa Moreno1, and Francielle Tramontini Gomes de Sousa Cardozo3
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1
Laboratory of Mutagenesis, Universidade Cato´lica Dom Bosco, Campo Grande – MS, Brazil, 2Laboratory of Applied Virology, Universidade Federal de Santa Catarina, Campus Universita´rio, Floriano´polis – SC, Brazil, and 3Laboratory of Virology, Universidade de Sa˜o Paulo, Instituto de Medicina Tropical, Sa˜o Paulo – SP, Brazil Abstract
Keywords
Context: Bothrops moojeni Hoge (Viperidae) venom is a complex mixture of compounds with therapeutic potential that has been included in the research and development of new drugs. Along with the biological activity, the pharmaceutical applicability of this venom depends on its toxicological profile. Objective: This study evaluates the cytotoxicity and genotoxicity of the Bothrops moojeni venom (BMV). Material and methods: The in vitro cytotoxicity and genotoxicity of a pooled sample of BMV was assessed by the MTT and Comet assay, respectively. Genotoxicity was also evaluated in vivo through the micronucleus assay. Results: BMV displayed a 50% cytotoxic concentration (CC50) on Vero cells of 4.09 mg/mL. Vero cells treated with 4 mg/mL for 90 min and 6 h presented significant (p50.05, ANOVA/Newman– Keuls test) higher DNA damage than the negative control in the Comet assay. The lower DNA damage found after 6 h compared with the 90 min treatment suggests a DNA repair effect. Mice intraperitoneally treated with BMV at 10, 30, or 80 mg/animal presented significant genotoxicity (p50.05, ANOVA/Newman–Keuls test) in relation to the negative control after 24 h of treatment. Contrary to the in vitro results, no DNA repair seemed to occur in vivo up to 96 h post-venom inoculation at a dose of 30 mg/animal. Discussion and conclusion: The results show that BMV presents cyto- and genotoxicity depending on the concentration/dose used. These findings emphasize the importance of toxicological studies, including assessment of genotoxicity, in the biological activity research of BMV and/or in the development of BMV-derived products.
Comet assay, cytotoxicity, genotoxicity, MTT assay, micronucleus assay
Introduction Snake venoms are a complex mixture of compounds responsible for local and systemic responses during envenoming, which is characterized by edema and inflammation (Galva˜o Nascimento et al., 2010), hemorrhage (De Roodt et al., 2003), hypotension (Silveira et al., 2013), nephrotoxicity (Barbosa et al., 2002), and myotoxicity (Santos-Filho et al., 2008). Despite their harmful effects, snake venoms represent an important source of bioactive molecules, contributing for the development of new therapeutic agents to be used in the treatment of envenomation and diseases such as cancer, thrombosis, and hypertension (Koh et al., 2006). Bothrops sp. venoms have been shown to have antiparasitic activity, against
Correspondence: Francielle Tramontini Gomes de Sousa Cardozo, Laboratory of Virology, Institute of Tropical Medicine, Universidade de Sa˜o Paulo (USP), Av Dr Eneas de Carvalho Aguiar, 470. Pre´dio 1, segundo andar, Cerqueira Ce´sar, Sa˜o Paulo – SP 05403000, Brazil. Tel: +55 11 3061 7020. Fax: +55 11 3061 8680. E-mail: [email protected]
History Received 13 August 2013 Revised 1 July 2014 Accepted 25 July 2014 Published online 27 November 2014
Leishmania spp. (Tempone et al., 2001) and Trypanosoma cruzi (Deolindo et al., 2005); antimicrobial effects on Pseudomonas aeruginosa, Candida albicans, Escherichia coli, and Staphylococcus aureus (Ciscotto et al., 2009; Rodrigues et al., 2004; Torres et al., 2010); and inhibitory effect on in vitro invasion and replication of Toxoplasma gondii (Bastos et al., 2008). Bothrops moojeni Hoge (Viperidae) is a large pit viper species, popularly known as ‘‘caic¸aca’’ or ‘‘jararaca˜o’’, mainly found in central and southeastern Brazil. Its venom has been attracting researchers’ attention since it is composed of a complex mixture of proteins presenting biological activity, and then it is a rich source of new drugs candidates (Oliveira Ju´nior et al., 2013; Paulchamy, 2010). Along with the biological activity evaluation, nonclinical safety assessment is an essential step on the development of new therapeutic agents from snake venoms, providing prospective data on their safety. In this sense, the aim of this study was to evaluate the cytotoxicity and genotoxicity of the Bothrops moojeni venom using in vitro and in vivo methods.
DOI: 10.3109/13880209.2014.950385
Materials and methods A pooled sample of Bothrops moojeni venom (BMV) was obtained from adult specimens, originated from Campo Grande, Mato Grosso do Sul, Brazil, and kept in captivity in the serpentarium of the Universidade Cato´lica Dom Bosco (UCDB), lyophilized, filtered (0.22 mm, Millipore, Billerica, MA), and kept at 20 C until use.
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Evaluation of cytotoxicity The cytotoxicity of BMV was determined by the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Mosmann, 1983). Briefly, Vero cells (CCL81; ATCC, Rockville, MD) were seeded in 96-well plates (2.5 105 cells/ mL) and grown in minimal essential medium (MEM), supplemented with 10% fetal bovine serum and antibiotics, in an atmosphere of 5% CO2 at 37 C for 24 h. Subsequently, cells monolayers were treated with different concentrations of BMV, at 1:2 serial dilutions ranging from 200 to 0.195 mg/mL. After 48 h, the medium was removed, 50 mL of MTT solution (1 mg/mL) was added, and plates were reincubated for 4 h. The MTT solution was removed and 100 mL of DMSO was added to dissolve formazan crystals. Afterward, absorbances were read on a multiwell spectrophotometer (Bio-Tek, Winooski, VT, Elx 800) at 540 nm and values were used to build concentration–response curves. The 50% cytotoxic concentration (CC50) was calculated as the concentration that reduces cell viability by 50%, when compared with the untreated controls. Evaluation of the in vitro genotoxicity Genotoxicity was evaluated by the alkaline Comet assay, performed as described by Singh et al. (1988), with some modifications according to International Conference on Harmonization (ICH, 2012) and Klaude et al. (1996). In brief, Vero cells (2.5 105 cells/mL), grown in 24-well tissue culture plates, were treated with BMV at three different concentrations (1, 2, or 4 mg/mL) for 90 min or 6 h. Positive (200 mM H2O2) and negative (only MEM) controls were included in each experiment. After treatment, Vero cells were washed three times with phosphate-buffered saline (PBS, pH 7.4), trypsinized, centrifuged, and resuspended in MEM to reach a density of 1 106 cells/mL. Then, cell suspensions were mixed with low-melting point agarose, spread on agarose-precoated slides, and cooled to allow the cell agar layer to solidify. Then, coverslip was gently removed and the slides were submerged into an ice-cold lysing solution (2.5 M NaCl, 10 mM Tris, 0.1 mM EDTA, 1% sodium sarcosinate, 1% Triton X-100, and 10% DMSO; pH 10) at 4 C for at least 1 h. Slides were subsequently incubated in alkaline buffer (300 mM NaOH and 1 mM EDTA, pH 413) for 30 min, at 4 C. Electrophoresis was performed at a fixed voltage (25 V) and current between 280 and 300 mA for 30 min. Slides were neutralized into Tris-HCl buffer (pH 7.5) for 5 min. After staining with 50 mL of ethidium bromide (20 mg/mL), slides were analyzed under an epifluorescence microscopy (Olympus BX 40, Olympus, Center Valley, PA, 515–560 nm excitation filter and a 590 nm barrier filter) at 400-fold magnification. Fifty fluorescent-stained nucleoids from the
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central area of each slide (two slides per concentration) were scored into five categories, according to the following score system: 0, no damaged, nucleoids with no tail; 1, comets with small tails (tail length less than a quarter of head diameter); 2, comets with medium tails (tail length between a quarter and a full of head diameter); 3, comets with long tails (tail length greater than head diameter); 4, comets with poorly defined or small head. Damage index was calculated by summing the scores of 50 comets. Evaluation of the in vivo genotoxicity All the in vivo experiments were approved by UCDB’s Ethics Committee for research on animals (approval number 010/08) and this work was performed according to national and international ethical guidelines in the experiments involving the use of laboratory animals. Male Swiss mice, obtained from the bioterium of UCDB, weighing 18–22 g, were used in two experimental protocols. Protocol A (different doses evaluated at the same time period) Different groups of seven animals were treated intraperitoneally with 200 mL of BMV solution diluted in PBS, at 10, 30, or 80 mg/animal (50, 150, and 400 mg/mL, respectively). Positive and negative control groups were treated with cyclophosphamide at 50 mg/kg and PBS, respectively. Blood samples (200 mL) were collected 24 h after treatments, from dorsal pedal vein, with no anesthesia and using a 25-Gauge needle, according to the procedures described by Parasuraman et al. (2010). Protocol B (same dose evaluated at different time periods) Groups of seven animals were treated intraperitoneally with 200 mL of BMV solution at 30 mg/animal (150 mg/mL). Positive and negative control groups were treated with colchicine (0.5 mg/kg) and PBS, respectively. Blood samples were collected at 24, 48, 72, and 96 h after dosing. Peripheral blood erythrocytes were evaluated through micronucleus assay as described previously (Chieco & Derenzini, 1999; Hayashi et al., 2000; Schmid, 1975), with minor modifications. Briefly, blood samples were dropped onto slides, airdried overnight, fixed with absolute methanol for 10 min, stained with Schiff’s reagent for 60 min, counterstained with Fast Green 1% for 1 min, and mounted with coverslips. Slides were observed under a fluorescent microscope and the incidence of micronuclei was determined based on observation of 1000 cells per sample. Statistical analysis The CC50 value was estimated by linear regression analysis of concentration–response curves. The results were expressed as mean ± SEM of three independent experiments. Treated groups were compared with the positive and negative controls by analysis of variance (one-way ANOVA) followed by Newman–Keuls multiple comparison test using GraphPad Prism software (GraphPad, San Diego, CA) (version 5.01). Values of p50.05 were considered statistically significant.
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Results
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BMV presented a concentration-dependent cytotoxicity with Vero cells and the CC50 obtained value was 4.09 ± 0.04 mg/mL. In vitro genotoxicity evaluation (Figure 1) showed that BMV displayed significant higher DNA damages than the negative control, when tested at 4 mg/mL, in both treatment periods tested (90 min and 6 h). All doses tested showed significant differences compared with the positive control group.
Figure 1. In vitro evaluation of Bothrops moojeni venom (BMV) genotoxicity by the Comet assay. Vero cells were treated with different concentrations of BMV (1, 2, and 4 mg/mL) for 90 min or 6 h. Fifty fluorescent-stained nucleoids from the central area of each slide were scored into five types, according to the score system described in Material and methods section. Positive and negative controls were treated with 200 mM H2O2 and MEM, respectively. Results were expressed as mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences (ANOVA followed by the Newman–Keuls multiple comparison test) in relation to the controls with p50.05.
In the present study, the genotoxicity was also evaluated in vivo. Swiss mice intraperitoneally treated with 10, 30, or 80 mg/animal of BMV showed a significant increase in micronucleus frequency, in blood samples collected after 24 h of exposure, in comparison with the negative control (Figure 2A). At the higher dose tested (80 mg/animal), BMV displayed genotoxicity (4.50 ± 1.29) statistically equivalent to the positive control (cyclophosphamide, 4.75 ± 0.43) and induced death in two animals 24 h post-treatment. Figure 2(B) shows that mice treated with 30 mg/animal of BMV presented significant higher micronucleus frequency than the negative control and the values were similar until 96 h post-BMV administration.
Discussion Many compounds isolated from BMV have been historically included in the research and development of new drugs. For example, Barbosa et al. (2010) reported the in vitro antibacterial and antiplatelet aggregation activities, the insulin secretion stimulation, and the in vivo improvement of kidney function for a lectin-like protein isolated from BMV. Morais et al. (2012) showed the anticoagulant and fibrinogenolytic activities of moojenin, a metalloproteinase extracted from BMV. Recently, an acidic phospholipase A2 from BMV was also shown to have bactericidal and anti-platelet
Figure 2. In vivo evaluation of Bothrops moojeni venom (BMV) genotoxicity by the micronucleus assay. (A) Peripheral blood reticulocytes from Swiss mice (n ¼ 7) intraperitoneally treated with BMV (10, 30, or 80 mg/animal; 50, 150, or 400 mg/mL, respectively) were evaluated at 24 h after dosing. Positive (PC) and negative (NC) control groups were treated with cyclophosphamide at 50 mg/kg and PBS, respectively. (B) Peripheral blood reticulocytes from Swiss mice (n ¼ 7) intraperitoneally treated with BMV (30 mg/animal, 150 mg/mL) were evaluated 24, 48, 72, and 96 h after dosing. Positive and negative control groups were treated with colchicine (0.5 mg/kg) and PBS, respectively. The frequency of micronucleus was expressed per 1000 cells as mean ± SEM. Asterisks indicate statistically significant differences (ANOVA followed by the Newman–Keuls multiple comparison test) in relation to the controls with p50.05.
aggregation effects, along with phospholipase activity and a moderated dose-dependent cytotoxic activity against tumor cell lines (Jukart, SKBR3, and EAT) (Silveira et al., 2013). Batroxobin, a serine protease derived from Bothrops atrox and B. moojeni venoms (Yonamine et al., 2013), presents thrombin-like activity and is commercially available for pharmaceutical and diagnostic applications (Perchuc et al., 2006). Diverse pharmacological activities have also been described for the crude venom of B. moojeni (BMV). For example, leishmanicidal activity (Castilhos et al., 2011), including a selective activity – a significant effect against promastigotes with no action on amastigotes forms (Tempone et al., 2001). BMV presented an antibacterial effect against Streptococcus mutans, the main etiological species of biofilms in dental caries (Mosca & Nascimento, 2011). BMV have also displayed peripheral antinociceptive activity in Swiss mice treated intraperitoneally (Rebouc¸as, 2010).
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DOI: 10.3109/13880209.2014.950385
Although isolated compounds from snake venoms are more likely to be developed as novel therapeutic agents, this study brings out the fact that BMV may present cyto- and genotoxicity depending on the concentration/dose used and this emphasizes the importance of toxicological studies, including assessment of genotoxicity, in the biological activity research of BMV crude preparations and/or in the development of BMV derived bioproducts. The selection of venom concentrations to be used in the in vitro Comet assay was based on the preliminary cytotoxicity evaluation (CC50 ¼ 4.09 ± 0.04 mg/mL). Since evaluation of genotoxicity should be performed at a concentration which do not exceed the CC50 (ICH, 2012), 4, 2, and 1 mg/mL had been selected for the Comet assay. Results presented in Figure 1 show that BMV was genotoxic to Vero cells at 4 mg/ mL. Moreover, cells treated with different concentrations of BMV (4, 2, or 1 mg/mL) or H2O2 (positive control) for 90 min had higher damage frequency than those treated for 6 h. This effect might be associated with cellular DNA repair mechanisms, as described by Purschke et al. (2002), who observed that human fibroblasts displayed repair ability of hydrogen peroxide-induced DNA damage. Differing from our results, Marcussi et al. (2013) found no significant genotoxicity for BMV at 7.5 mg/mL by the Comet assay. The diverse obtained results might be related to the differences in the experimental parameters. Marcussi et al. (2013) used a different cell line (human lymphocytes) incubated with the venom for 4 h. In this work, we analyzed the in vitro genotoxicity in Vero cells treated for 90 min and 6 h. In the present work, the genotoxicity of BMV was confirmed by in vivo studies. Genotoxicity was significantly higher in treated animal groups (10, 30, and 80 mg/animal) in comparison with the negative control group (PBS treated) (Figure 2A). At a dose of 80 mg/animal, BMV induced death in two animals 24 h post-inoculation. Likewise, Swiss male mice injected intradermically with BMV presented a LD50 of approximately 128 mg/mL in a study performed by Vale et al. (2008). Zamune´r et al. (2004) also showed the lethality of BMV when administered by intramuscular injection, in chicks at a dose of approximately 90 mg/animal. The mortality rate (2/7) in the group treated with 80 mg/ animal led us to select the dose of 30 mg/animal for the subsequent in vivo testing, according to the procedure described in the guideline for genotoxicity evaluation (ICH, 2012). Figure 2(B) shows that different from the in vitro results, no reduction of genotoxicity was observed up to 96 h post-treatment. It is possible that more than one component may be involved in the BMV-detected genotoxicity. BMV was shown to present glycoproteins as L-amino acid oxidase (Ribeiro et al., 2007), which catalyzes the oxidative deamination of L-amino acids, producing a-keto acids, ammonia, and hydrogen peroxide, the latter being an important mediator of oxidative stress and a potent mutagen (Konat, 2003). BMVinduced inflammatory mediators, such as nitric oxide (NO) (Nascimento et al., 2010) might also be responsible for the genotoxic effect herein observed, since NO reacts with superoxide or oxygen, producing deleterious effects on DNA (Wu et al., 2006). BMV metalloproteinases may also be involved. For instance, Rucavado et al. (2002) showed that
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metalloproteinases from the Bothrops asper venom stimulate the release of cytokines such as TNF-a, IL-1b, or IL-6. Bothrops jararaca and B. atrox venoms promoted the release of TNF-a, IL-1b, IL-6, IL-10, and IFN-g (Petricevich et al., 2000; Zamune´r et al., 2004). IFN-g and TNF-a were demonstrated to have a dose-dependent genotoxic effect in human peripheral blood lymphocyte cultures (Lazutka, 1996). Nevertheless, the component(s) of BMV responsible for its genotoxicity herein demonstrated remains to be determined.
Conclusions In summary, the treatment of Vero cells and mice with BMV resulted in concentration-dependent cyto- and genotoxicity. BMV treatments promoted significant in vitro DNA damages at 4 mg/mL and increased the in vivo micronucleus frequency at the tested doses (10, 30, and 80 mg/animal). Although a reduction in the frequency and rate of in vitro DNA damages has been observed with the increasing incubation time, possibly related to a repair mechanism, this was not observed in vivo, at least at the tested dose (30 mg/animal) until 96 h after dosing. Further studies of fractionated components from BMV must be carried out in order to identify which compound(s) is/are responsible for the BMV toxic effects herein described.
Declaration of interest The authors declare that there are no conflicts of interest in this work. This study received financial support from CAPES/ PROCAD, NF 2007. F. T. G. S. C. is indebted to CNPq for the research fellowship (Process number 151180/2013-0).
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SHORT COMMUNICATION
In vitro and in vivo genotoxic evaluation of Bothrops moojeni snake venom Nathalia Novak Zobiole1, Thiago Caon2, Je´ssica Wildgrube Bertol2, Cintia Alves de Souza Pereira1, Brunna Mary Okubo1, Susana Elisa Moreno1, and Francielle Tramontini Gomes de Sousa Cardozo3
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1
Laboratory of Mutagenesis, Universidade Cato´lica Dom Bosco, Campo Grande – MS, Brazil, 2Laboratory of Applied Virology, Universidade Federal de Santa Catarina, Campus Universita´rio, Floriano´polis – SC, Brazil, and 3Laboratory of Virology, Universidade de Sa˜o Paulo, Instituto de Medicina Tropical, Sa˜o Paulo – SP, Brazil Abstract
Keywords
Context: Bothrops moojeni Hoge (Viperidae) venom is a complex mixture of compounds with therapeutic potential that has been included in the research and development of new drugs. Along with the biological activity, the pharmaceutical applicability of this venom depends on its toxicological profile. Objective: This study evaluates the cytotoxicity and genotoxicity of the Bothrops moojeni venom (BMV). Material and methods: The in vitro cytotoxicity and genotoxicity of a pooled sample of BMV was assessed by the MTT and Comet assay, respectively. Genotoxicity was also evaluated in vivo through the micronucleus assay. Results: BMV displayed a 50% cytotoxic concentration (CC50) on Vero cells of 4.09 mg/mL. Vero cells treated with 4 mg/mL for 90 min and 6 h presented significant (p50.05, ANOVA/Newman– Keuls test) higher DNA damage than the negative control in the Comet assay. The lower DNA damage found after 6 h compared with the 90 min treatment suggests a DNA repair effect. Mice intraperitoneally treated with BMV at 10, 30, or 80 mg/animal presented significant genotoxicity (p50.05, ANOVA/Newman–Keuls test) in relation to the negative control after 24 h of treatment. Contrary to the in vitro results, no DNA repair seemed to occur in vivo up to 96 h post-venom inoculation at a dose of 30 mg/animal. Discussion and conclusion: The results show that BMV presents cyto- and genotoxicity depending on the concentration/dose used. These findings emphasize the importance of toxicological studies, including assessment of genotoxicity, in the biological activity research of BMV and/or in the development of BMV-derived products.
Comet assay, cytotoxicity, genotoxicity, MTT assay, micronucleus assay
Introduction Snake venoms are a complex mixture of compounds responsible for local and systemic responses during envenoming, which is characterized by edema and inflammation (Galva˜o Nascimento et al., 2010), hemorrhage (De Roodt et al., 2003), hypotension (Silveira et al., 2013), nephrotoxicity (Barbosa et al., 2002), and myotoxicity (Santos-Filho et al., 2008). Despite their harmful effects, snake venoms represent an important source of bioactive molecules, contributing for the development of new therapeutic agents to be used in the treatment of envenomation and diseases such as cancer, thrombosis, and hypertension (Koh et al., 2006). Bothrops sp. venoms have been shown to have antiparasitic activity, against
Correspondence: Francielle Tramontini Gomes de Sousa Cardozo, Laboratory of Virology, Institute of Tropical Medicine, Universidade de Sa˜o Paulo (USP), Av Dr Eneas de Carvalho Aguiar, 470. Pre´dio 1, segundo andar, Cerqueira Ce´sar, Sa˜o Paulo – SP 05403000, Brazil. Tel: +55 11 3061 7020. Fax: +55 11 3061 8680. E-mail: [email protected]
History Received 13 August 2013 Revised 1 July 2014 Accepted 25 July 2014 Published online 27 November 2014
Leishmania spp. (Tempone et al., 2001) and Trypanosoma cruzi (Deolindo et al., 2005); antimicrobial effects on Pseudomonas aeruginosa, Candida albicans, Escherichia coli, and Staphylococcus aureus (Ciscotto et al., 2009; Rodrigues et al., 2004; Torres et al., 2010); and inhibitory effect on in vitro invasion and replication of Toxoplasma gondii (Bastos et al., 2008). Bothrops moojeni Hoge (Viperidae) is a large pit viper species, popularly known as ‘‘caic¸aca’’ or ‘‘jararaca˜o’’, mainly found in central and southeastern Brazil. Its venom has been attracting researchers’ attention since it is composed of a complex mixture of proteins presenting biological activity, and then it is a rich source of new drugs candidates (Oliveira Ju´nior et al., 2013; Paulchamy, 2010). Along with the biological activity evaluation, nonclinical safety assessment is an essential step on the development of new therapeutic agents from snake venoms, providing prospective data on their safety. In this sense, the aim of this study was to evaluate the cytotoxicity and genotoxicity of the Bothrops moojeni venom using in vitro and in vivo methods.
DOI: 10.3109/13880209.2014.950385
Materials and methods A pooled sample of Bothrops moojeni venom (BMV) was obtained from adult specimens, originated from Campo Grande, Mato Grosso do Sul, Brazil, and kept in captivity in the serpentarium of the Universidade Cato´lica Dom Bosco (UCDB), lyophilized, filtered (0.22 mm, Millipore, Billerica, MA), and kept at 20 C until use.
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Evaluation of cytotoxicity The cytotoxicity of BMV was determined by the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Mosmann, 1983). Briefly, Vero cells (CCL81; ATCC, Rockville, MD) were seeded in 96-well plates (2.5 105 cells/ mL) and grown in minimal essential medium (MEM), supplemented with 10% fetal bovine serum and antibiotics, in an atmosphere of 5% CO2 at 37 C for 24 h. Subsequently, cells monolayers were treated with different concentrations of BMV, at 1:2 serial dilutions ranging from 200 to 0.195 mg/mL. After 48 h, the medium was removed, 50 mL of MTT solution (1 mg/mL) was added, and plates were reincubated for 4 h. The MTT solution was removed and 100 mL of DMSO was added to dissolve formazan crystals. Afterward, absorbances were read on a multiwell spectrophotometer (Bio-Tek, Winooski, VT, Elx 800) at 540 nm and values were used to build concentration–response curves. The 50% cytotoxic concentration (CC50) was calculated as the concentration that reduces cell viability by 50%, when compared with the untreated controls. Evaluation of the in vitro genotoxicity Genotoxicity was evaluated by the alkaline Comet assay, performed as described by Singh et al. (1988), with some modifications according to International Conference on Harmonization (ICH, 2012) and Klaude et al. (1996). In brief, Vero cells (2.5 105 cells/mL), grown in 24-well tissue culture plates, were treated with BMV at three different concentrations (1, 2, or 4 mg/mL) for 90 min or 6 h. Positive (200 mM H2O2) and negative (only MEM) controls were included in each experiment. After treatment, Vero cells were washed three times with phosphate-buffered saline (PBS, pH 7.4), trypsinized, centrifuged, and resuspended in MEM to reach a density of 1 106 cells/mL. Then, cell suspensions were mixed with low-melting point agarose, spread on agarose-precoated slides, and cooled to allow the cell agar layer to solidify. Then, coverslip was gently removed and the slides were submerged into an ice-cold lysing solution (2.5 M NaCl, 10 mM Tris, 0.1 mM EDTA, 1% sodium sarcosinate, 1% Triton X-100, and 10% DMSO; pH 10) at 4 C for at least 1 h. Slides were subsequently incubated in alkaline buffer (300 mM NaOH and 1 mM EDTA, pH 413) for 30 min, at 4 C. Electrophoresis was performed at a fixed voltage (25 V) and current between 280 and 300 mA for 30 min. Slides were neutralized into Tris-HCl buffer (pH 7.5) for 5 min. After staining with 50 mL of ethidium bromide (20 mg/mL), slides were analyzed under an epifluorescence microscopy (Olympus BX 40, Olympus, Center Valley, PA, 515–560 nm excitation filter and a 590 nm barrier filter) at 400-fold magnification. Fifty fluorescent-stained nucleoids from the
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central area of each slide (two slides per concentration) were scored into five categories, according to the following score system: 0, no damaged, nucleoids with no tail; 1, comets with small tails (tail length less than a quarter of head diameter); 2, comets with medium tails (tail length between a quarter and a full of head diameter); 3, comets with long tails (tail length greater than head diameter); 4, comets with poorly defined or small head. Damage index was calculated by summing the scores of 50 comets. Evaluation of the in vivo genotoxicity All the in vivo experiments were approved by UCDB’s Ethics Committee for research on animals (approval number 010/08) and this work was performed according to national and international ethical guidelines in the experiments involving the use of laboratory animals. Male Swiss mice, obtained from the bioterium of UCDB, weighing 18–22 g, were used in two experimental protocols. Protocol A (different doses evaluated at the same time period) Different groups of seven animals were treated intraperitoneally with 200 mL of BMV solution diluted in PBS, at 10, 30, or 80 mg/animal (50, 150, and 400 mg/mL, respectively). Positive and negative control groups were treated with cyclophosphamide at 50 mg/kg and PBS, respectively. Blood samples (200 mL) were collected 24 h after treatments, from dorsal pedal vein, with no anesthesia and using a 25-Gauge needle, according to the procedures described by Parasuraman et al. (2010). Protocol B (same dose evaluated at different time periods) Groups of seven animals were treated intraperitoneally with 200 mL of BMV solution at 30 mg/animal (150 mg/mL). Positive and negative control groups were treated with colchicine (0.5 mg/kg) and PBS, respectively. Blood samples were collected at 24, 48, 72, and 96 h after dosing. Peripheral blood erythrocytes were evaluated through micronucleus assay as described previously (Chieco & Derenzini, 1999; Hayashi et al., 2000; Schmid, 1975), with minor modifications. Briefly, blood samples were dropped onto slides, airdried overnight, fixed with absolute methanol for 10 min, stained with Schiff’s reagent for 60 min, counterstained with Fast Green 1% for 1 min, and mounted with coverslips. Slides were observed under a fluorescent microscope and the incidence of micronuclei was determined based on observation of 1000 cells per sample. Statistical analysis The CC50 value was estimated by linear regression analysis of concentration–response curves. The results were expressed as mean ± SEM of three independent experiments. Treated groups were compared with the positive and negative controls by analysis of variance (one-way ANOVA) followed by Newman–Keuls multiple comparison test using GraphPad Prism software (GraphPad, San Diego, CA) (version 5.01). Values of p50.05 were considered statistically significant.
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Results
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BMV presented a concentration-dependent cytotoxicity with Vero cells and the CC50 obtained value was 4.09 ± 0.04 mg/mL. In vitro genotoxicity evaluation (Figure 1) showed that BMV displayed significant higher DNA damages than the negative control, when tested at 4 mg/mL, in both treatment periods tested (90 min and 6 h). All doses tested showed significant differences compared with the positive control group.
Figure 1. In vitro evaluation of Bothrops moojeni venom (BMV) genotoxicity by the Comet assay. Vero cells were treated with different concentrations of BMV (1, 2, and 4 mg/mL) for 90 min or 6 h. Fifty fluorescent-stained nucleoids from the central area of each slide were scored into five types, according to the score system described in Material and methods section. Positive and negative controls were treated with 200 mM H2O2 and MEM, respectively. Results were expressed as mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences (ANOVA followed by the Newman–Keuls multiple comparison test) in relation to the controls with p50.05.
In the present study, the genotoxicity was also evaluated in vivo. Swiss mice intraperitoneally treated with 10, 30, or 80 mg/animal of BMV showed a significant increase in micronucleus frequency, in blood samples collected after 24 h of exposure, in comparison with the negative control (Figure 2A). At the higher dose tested (80 mg/animal), BMV displayed genotoxicity (4.50 ± 1.29) statistically equivalent to the positive control (cyclophosphamide, 4.75 ± 0.43) and induced death in two animals 24 h post-treatment. Figure 2(B) shows that mice treated with 30 mg/animal of BMV presented significant higher micronucleus frequency than the negative control and the values were similar until 96 h post-BMV administration.
Discussion Many compounds isolated from BMV have been historically included in the research and development of new drugs. For example, Barbosa et al. (2010) reported the in vitro antibacterial and antiplatelet aggregation activities, the insulin secretion stimulation, and the in vivo improvement of kidney function for a lectin-like protein isolated from BMV. Morais et al. (2012) showed the anticoagulant and fibrinogenolytic activities of moojenin, a metalloproteinase extracted from BMV. Recently, an acidic phospholipase A2 from BMV was also shown to have bactericidal and anti-platelet
Figure 2. In vivo evaluation of Bothrops moojeni venom (BMV) genotoxicity by the micronucleus assay. (A) Peripheral blood reticulocytes from Swiss mice (n ¼ 7) intraperitoneally treated with BMV (10, 30, or 80 mg/animal; 50, 150, or 400 mg/mL, respectively) were evaluated at 24 h after dosing. Positive (PC) and negative (NC) control groups were treated with cyclophosphamide at 50 mg/kg and PBS, respectively. (B) Peripheral blood reticulocytes from Swiss mice (n ¼ 7) intraperitoneally treated with BMV (30 mg/animal, 150 mg/mL) were evaluated 24, 48, 72, and 96 h after dosing. Positive and negative control groups were treated with colchicine (0.5 mg/kg) and PBS, respectively. The frequency of micronucleus was expressed per 1000 cells as mean ± SEM. Asterisks indicate statistically significant differences (ANOVA followed by the Newman–Keuls multiple comparison test) in relation to the controls with p50.05.
aggregation effects, along with phospholipase activity and a moderated dose-dependent cytotoxic activity against tumor cell lines (Jukart, SKBR3, and EAT) (Silveira et al., 2013). Batroxobin, a serine protease derived from Bothrops atrox and B. moojeni venoms (Yonamine et al., 2013), presents thrombin-like activity and is commercially available for pharmaceutical and diagnostic applications (Perchuc et al., 2006). Diverse pharmacological activities have also been described for the crude venom of B. moojeni (BMV). For example, leishmanicidal activity (Castilhos et al., 2011), including a selective activity – a significant effect against promastigotes with no action on amastigotes forms (Tempone et al., 2001). BMV presented an antibacterial effect against Streptococcus mutans, the main etiological species of biofilms in dental caries (Mosca & Nascimento, 2011). BMV have also displayed peripheral antinociceptive activity in Swiss mice treated intraperitoneally (Rebouc¸as, 2010).
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DOI: 10.3109/13880209.2014.950385
Although isolated compounds from snake venoms are more likely to be developed as novel therapeutic agents, this study brings out the fact that BMV may present cyto- and genotoxicity depending on the concentration/dose used and this emphasizes the importance of toxicological studies, including assessment of genotoxicity, in the biological activity research of BMV crude preparations and/or in the development of BMV derived bioproducts. The selection of venom concentrations to be used in the in vitro Comet assay was based on the preliminary cytotoxicity evaluation (CC50 ¼ 4.09 ± 0.04 mg/mL). Since evaluation of genotoxicity should be performed at a concentration which do not exceed the CC50 (ICH, 2012), 4, 2, and 1 mg/mL had been selected for the Comet assay. Results presented in Figure 1 show that BMV was genotoxic to Vero cells at 4 mg/ mL. Moreover, cells treated with different concentrations of BMV (4, 2, or 1 mg/mL) or H2O2 (positive control) for 90 min had higher damage frequency than those treated for 6 h. This effect might be associated with cellular DNA repair mechanisms, as described by Purschke et al. (2002), who observed that human fibroblasts displayed repair ability of hydrogen peroxide-induced DNA damage. Differing from our results, Marcussi et al. (2013) found no significant genotoxicity for BMV at 7.5 mg/mL by the Comet assay. The diverse obtained results might be related to the differences in the experimental parameters. Marcussi et al. (2013) used a different cell line (human lymphocytes) incubated with the venom for 4 h. In this work, we analyzed the in vitro genotoxicity in Vero cells treated for 90 min and 6 h. In the present work, the genotoxicity of BMV was confirmed by in vivo studies. Genotoxicity was significantly higher in treated animal groups (10, 30, and 80 mg/animal) in comparison with the negative control group (PBS treated) (Figure 2A). At a dose of 80 mg/animal, BMV induced death in two animals 24 h post-inoculation. Likewise, Swiss male mice injected intradermically with BMV presented a LD50 of approximately 128 mg/mL in a study performed by Vale et al. (2008). Zamune´r et al. (2004) also showed the lethality of BMV when administered by intramuscular injection, in chicks at a dose of approximately 90 mg/animal. The mortality rate (2/7) in the group treated with 80 mg/ animal led us to select the dose of 30 mg/animal for the subsequent in vivo testing, according to the procedure described in the guideline for genotoxicity evaluation (ICH, 2012). Figure 2(B) shows that different from the in vitro results, no reduction of genotoxicity was observed up to 96 h post-treatment. It is possible that more than one component may be involved in the BMV-detected genotoxicity. BMV was shown to present glycoproteins as L-amino acid oxidase (Ribeiro et al., 2007), which catalyzes the oxidative deamination of L-amino acids, producing a-keto acids, ammonia, and hydrogen peroxide, the latter being an important mediator of oxidative stress and a potent mutagen (Konat, 2003). BMVinduced inflammatory mediators, such as nitric oxide (NO) (Nascimento et al., 2010) might also be responsible for the genotoxic effect herein observed, since NO reacts with superoxide or oxygen, producing deleterious effects on DNA (Wu et al., 2006). BMV metalloproteinases may also be involved. For instance, Rucavado et al. (2002) showed that
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metalloproteinases from the Bothrops asper venom stimulate the release of cytokines such as TNF-a, IL-1b, or IL-6. Bothrops jararaca and B. atrox venoms promoted the release of TNF-a, IL-1b, IL-6, IL-10, and IFN-g (Petricevich et al., 2000; Zamune´r et al., 2004). IFN-g and TNF-a were demonstrated to have a dose-dependent genotoxic effect in human peripheral blood lymphocyte cultures (Lazutka, 1996). Nevertheless, the component(s) of BMV responsible for its genotoxicity herein demonstrated remains to be determined.
Conclusions In summary, the treatment of Vero cells and mice with BMV resulted in concentration-dependent cyto- and genotoxicity. BMV treatments promoted significant in vitro DNA damages at 4 mg/mL and increased the in vivo micronucleus frequency at the tested doses (10, 30, and 80 mg/animal). Although a reduction in the frequency and rate of in vitro DNA damages has been observed with the increasing incubation time, possibly related to a repair mechanism, this was not observed in vivo, at least at the tested dose (30 mg/animal) until 96 h after dosing. Further studies of fractionated components from BMV must be carried out in order to identify which compound(s) is/are responsible for the BMV toxic effects herein described.
Declaration of interest The authors declare that there are no conflicts of interest in this work. This study received financial support from CAPES/ PROCAD, NF 2007. F. T. G. S. C. is indebted to CNPq for the research fellowship (Process number 151180/2013-0).
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