EFFECT OF Crotalus durissus terrificus (LAURENTI, 1768) VENOM ON THE EVOLUTION OF EHRLICH ASCITES TUMOR

Abstract

The effect of Crotalus durissus terrificus (LAURENTI, 1768) venom on the evolution of Ehrlich ascites tumor cells was evaluated. Thus, 30-day-old male mice of the Swiss strain were inoculated intraperitoneally with 1x10<img SRC="http:/img/fbpe/jvat/v3n2/image1947.gif"> tumor cells. Then, 7 groups of animals were formed: 3 control groups (physiological, venom and tumor) and 4 experimental groups that received different doses of venom. The experimental groups received 5 intraperitoneal venom injections on the 1<img SRC="http:/img/fbpe/jvat/v3n2/image1948.gif"> , 4<img SRC="http:/img/fbpe/jvat/v3n2/image1949.gif"> , 7<img SRC="http:/img/fbpe/jvat/v3n2/image1949.gif"> , 10<img SRC="http:/img/fbpe/jvat/v3n2/image1949.gif"> and 13<img SRC="http:/img/fbpe/jvat/v3n2/image1949.gif"> days after tumor implantation. On the 14<img SRC="http:/img/fbpe/jvat/v3n2/image1949.gif" width="28" height="28"> day, 5 animals from each one of the groups were sacrificed, and the variables such as the total and differential counts of cells in the peritoneal cavity and functional state of peritoneal macrophages by macrophage spreading were evaluated. The other 5 remaining animals were kept in the laboratory for 60 days for observation of their survival percentage. The results obtained were statistically analyzed by the Kruskal-Wallis test at 5% significance level. It was observed that Crotalus durissus terrificus venom increases survival time of mice, but does not increase mortality percentage. This venom also increases the percentage of macrophage spreading. We suggest that snake venoms can cause inhibition of tumor growth by activating the inflammatory reaction, mainly the macrophages, stimulating the production of TNF-<img SRC="http:/img/fbpe/jvat/v3n2/image1950.gif"> , IL-1, IL-6 and IL-8. These cytokines may act on tumor cells by different mechanisms, inducing its complete elimination.

venom; inflammation; tumor growth; Crotalus durissus terrificus

Original paper

EFFECT OF Crotalus durissus terrificus (LAURENTI, 1768) VENOM ONTHE EVOLUTION OF EHRLICH ASCITES TUMOR

R. J. DA SILVA

, D. FECCHIO , B. BARRAVIERA

1 Center for the Study of Venoms and Venomous Animals - CEVAP-UNESP, State of São Paulo, Brazil; 2 Department of Pathology of the School of Medicine of Botucatu, State of São Paulo, Brazil, 3 Department of Tropical Diseases of the School of Medicine of Botucatu, State of São Paulo, Brazil.

ABSTRACT. The effect of Crotalus durissus terrificus (LAURENTI, 1768) venom on the evolution of Ehrlich ascites tumor cells was evaluated. Thus, 30-day-old male mice of the Swiss strain were inoculated intraperitoneally with 1x10 tumor cells. Then, 7 groups of animals were formed: 3 control groups (physiological, venom and tumor) and 4 experimental groups that received different doses of venom. The experimental groups received 5 intraperitoneal venom injections on the 1 , 4 , 7 , 10 and 13 days after tumor implantation. On the 14 day, 5 animals from each one of the groups were sacrificed, and the variables such as the total and differential counts of cells in the peritoneal cavity and functional state of peritoneal macrophages by macrophage spreading were evaluated. The other 5 remaining animals were kept in the laboratory for 60 days for observation of their survival percentage. The results obtained were statistically analyzed by the Kruskal-Wallis test at 5% significance level. It was observed that Crotalus durissus terrificus venom increases survival time of mice, but does not increase mortality percentage. This venom also increases the percentage of macrophage spreading. We suggest that snake venoms can cause inhibition of tumor growth by activating the inflammatory reaction, mainly the macrophages, stimulating the production of TNF- , IL-1, IL-6 and IL-8. These cytokines may act on tumor cells by different mechanisms, inducing its complete elimination.

KEY WORDS: venom, inflammation, tumor growth, Crotalus durissus terrificus.

INTRODUCTION

The search for antitumor agents has been pursued by a number of researchers. Several experimental studies using snake venoms in the treatment of tumor were carried out (3,5,6,7,8,9) (15,16,19,21,27,33). However, as we(29) have previously reported, controversies still exist. Some authors have reported that the treatment of tumor with certain venom fractions or with crude venom of some species of snakes have an important toxic effect on tumor cells. Complete elimination of tumor cells was obtained in some cases(19). On the other hand, other authors have not detected any antitumor effect(29).

In the above mentioned research(3,5,6,7,8,9)(15,16,19,21,27,33) venoms of different snake species were used. Snake venoms of the genera Naja and Crotalus were used with successful results. However, the mechanism involved in tumor elimination after treatment with snake venom is still unknown. It is well known in literature that certain tumors are capable of inhibiting the host inflammatory response (10,12,20,30,31). This mechanism can facilitate tumor development. On the contrary, as previously reported by Silva et al.(28), intense activation of the inflammatory response occurs when mice are inoculated with snake venom. Thus, determination of the parameters of the host inflammatory response in experimental models of tumor growth inhibition is fundamental.

Therefore, the objective of the present paper was to determine the effects of the treatment of animals bearing Ehrlich ascites tumor using Crotalus durissus terrificus venom. Thus, we analyzed not only tumor growth but also parameters of acute inflammatory response of inoculated animals, determining the effects of envenomation on polymorphonuclear (PMN) and mononuclear (MN) cells, mainly macrophages.

MATERIALS AND METHODS

ANIMALS: Thirty-day old, male albino mice of the swiss strains (Mus musculus), weighing from 20 to 30 grams were used. The animals were kept in cages and received water and food ad libitum. Wood chips were used as substrate.

VENOM: Crotalus durissus terrificus(2) venom was milked from snakes kept in the Serpentarium of The Center for the Study of Venoms and Venomous Animals (CEVAP) at São Paulo State University (UNESP), Botucatu, SP, Brazil. Immediately after milking, the venom was centrifuged for 10 min at 1,500 rpm. and filtered in Millipore GSWP00250 filter. The venom sample was lyophilized and stored in a refrigerator at 4°C throughout the experiment. The venom LD50 (24,25) was previously determined as 0.1 mg/kg of weight.

TUMOR: Cells of Ehrlich ascites tumor were used. After harvesting and preparation of the cells, their total number and viability were determined by counting in Neubauer chamber using Trypan blue(11). The desired concentration of tumor cells (1x10 cells per 0.1 ml) was obtained by dilution with saline (0.9% sodium chloride solution). Viability of tumor cells obtained and used in this experiment was always higher than 90.0%. Below this percentage, the cells were discarded and the entire procedure was repeated.

COUNT OF THE TOTAL NUMBER OF CELLS PRESENT IN THE PERITONEAL CAVITY: The total number of cells in the peritoneal cavity was determined by counting in Neubauer chamber(11).

DIFFERENTIAL COUNT OF CELLS PRESENT IN THE PERITONEAL CAVITY: The cells in the peritoneal cavity of mice were harvested and stained and later differentiated into MN, PMN and tumor cells. To differentiate MN from PMN cells, classical morphological patterns(18) were used. To identify tumor cells, the paper by Fecchio(11) was used as basic reference.

DETERMINATION OF FUNCTIONAL ACTIVITY OF MACROPHAGES: Functional activity of macrophages in the peritoneal cavity was determined by the spreading technique adapted from that by Rabinovitch et al. (23). Thus, 0.1 ml of the cellular suspension obtained in the peritoneal cavity was placed over glass coverslips at room temperature for 15 min. The non-adherent cells were removed by washing with phosphate buffer saline, and the adherent cells were incubated in culture medium 199 containing 10 nM Hepes at 37°C for 1 h. Following this, the culture medium was removed and the cells were fixed with 2.5% glutaraldehyde. Then, the cells were stained with Giemsa and examined under microscopy where the percentage of spread cells was determined under 40x magnification. Spread cells were those that presented cytoplamic elongation, while the non-spread cells were rounded (11,22,23) (Figure 6).

EXPERIMENTAL DESIGN: Seven groups of 10 mice were formed, as follows:

G1 - saline control;

G2 - venom control (1.6 µg/kg);

G3 - tumor control (EAT);

G4 - EAT + 0.2 µg/kg of venom;

G5 - EAT + 0.4 µg/kg of venom;

G6 - EAT + 0.8 µg/kg of venom;

G7 - EAT + 1.6 µg/kg of venom.

The animals' treatment began 24 hours after tumor inoculation. Treatment with venom consisted of 5 intraperitoneal injections with a 72-hour interval between each inoculation. The venom control group (G2) was injected only with venom, while the saline control group (G1) and tumor control group (G3) were injected only with saline solution. The other remaining groups (G4, G5, G6 and G7) were injected with different doses of the venom. The next day after the last inoculation, i.e., on the 14 day after tumor inoculation, 5 animals of each group were chosen by lot to be sacrificed. The animals were sacrificed in sulphure ether chamber. After asepsis of abdominal region, each animal was inoculated with 3 ml of saline solution. After homogenization, the solution containing peritoneal cells was removed for cellular evaluation. The following variables were analyzed:

- the total number of cells present in the peritoneal cavity;

- the percentage of tumor cells, MN and PMN cells;

- the functional activity of peritoneal macrophages by the spreading technique.

The remaining animals, i.e., 5 animals of each group studied were kept in the laboratory for 60 days when their survival percentage was evaluated. These procedures were repeated 3 times, and data were grouped for statistical analysis

STATISTICAL ANALYSIS: Since the variables analyzed did not show normal distribution, data obtained were analyzed by the non-parametric Kruskal-Wallis test, and the difference among the groups was determined by Tuckey's test(34). The significance level was 5%.

RESULTS

DETERMINATION OF TOTAL NUMBER OF CELLS PRESENT IN THE PERITONEAL CAVITY: The results obtained for the variable total number of cells in the peritoneal cavity of the animals are shown in Table 1 and Figure 1. Of all the experimental groups studied, the tumor control group (G3) presented higher quantity of cells in the peritoneal cavity. The tumor-bearing groups treated with Crotalus durissus terrificus venom (G4, G5, G6 and G7) showed lower medians for this variable than that of the tumor control group (G3). However, these groups did not present a statistically significant difference in relation to the tumor control group (G3). In addition, all these groups (G3, G4, G5, G6 and G7) showed significantly higher values than those of the saline (G1) and the venom (G2) control groups. There was no significant difference between the saline (G1) and the venom (G2) control groups. Likewise, although the statistical test has shown a marked tendency towards significance, no significant difference was observed between the venom control group (G2) and two groups that were inoculated with tumor and were treated with venom (G5 and G7).

TABLE 1.
Descriptive statistics for the variables observed for each experimental group on the 14

FIGURE 1.
Distribution of the total number of cells (N x 10

DETERMINATION OF THE TOTAL NUMBER OF TUMOR CELLS IN THE PERITONEAL CAVITY: The results obtained for the variable number of tumor cells in the peritoneal cavity of the animals are shown in Table 1 and in Figure 2. All the tumor-bearing groups (G3, G4, G5, G6 and G7) treated or non-treated with Crotalus durissus terrificus venom were statistically higher than the saline control (G1) and venom control (G2) groups. However, similarly to the total count of cells (Figure 1), the medians of all tumor-bearing groups treated with venom (G4, G5, G6 and G7), regardless of the dose, were lower than those of the tumor control group (G3). In spite of this, statistically significant differences were not observed between the tumor-bearing group non-treated with venom (G3) and the tumor-bearing group treated with Crotalus durissus terrificus venom (G4, G5, G6 and G7).

FIGURE 2.
Distribution of the number of tumor cells (Nx10

DETERMINATION OF THE NUMBER OF PMN CELLS IN THE PERITONEAL CAVITY: The results obtained for the variable number of PMN cells in the peritoneal cavity of animals are shown in Table 1 and in Figure 3. Both the tumor-bearing group without treatment (G3) and the groups treated with venom (G4, G5, G6, and G7) revealed significantly higher quantities of cells when compared with that of the saline control group (G1). There was no statistically significant difference between the saline (G1) and the venom (G2) control groups. Likewise, the same fact was observed when comparing the venom control group (G2) with the tumor-inoculated groups, either the treated (G4, G5, G6 and G7) or the non-treated (G3) groups.

FIGURE 3.
Distribution of the number of PMN cells (Nx10

DETERMINATION OF THE NUMBER OF MN CELLS IN THE PERITONEAL CAVITY: The results obtained for the variable number of MN cells in the peritoneal cavity of the animals are shown in Table 1 and in Figure 4. There was no statistically significant difference among all the experimental groups. The presence of the tumor as well as the treatment with Crotalus durissus terrificus venom did not produce alteration in the number of MN cells in the peritoneal cavity of the animals.

FIGURE 4.
Distribution of the number of MN cells (Nx10

DETERMINATION OF THE PERCENTAGE OF SPREADING OF MACROPHAGES HARVESTED IN THE PERITONEAL CAVITY: The results obtained for the variable percentage of spreading of macrophages harvested in the peritoneal cavity of the animals are shown in Table 1 and in Figure 5 and Figure 6. The treatment with Crotalus durissus terrificus venom yielded an increase in the functional activity of macrophages of normal animals only treated with venom (G2) and in two of the tumor-inoculated groups treated with venom (G6 and G7). These results when compared with the saline control group (G1) were statistically significant. The presence of tumor cells did not increase the percentage of macrophage spreading. This fact can be observed in the tumor control group (G3) which did not differ significantly from the saline control group (G1). It should also be pointed out that for the tumor-bearing groups treated with venom (G4, G5, G6 and G7), there was an increase in the median value of spreading percentage accompanying the increase in the venom dose. However, the difference among such values was not statistically significant.

FIGURE 5.
Distribution of the percentage of macrophage spreading in the peritoneal cavity of the animals on the 14

FIGURE 6.
Macrophage spreading determined from ascitic fluid obtained from: A, EAT control group (G3) and B, EAT + 1.6 mg/kg of Crotalus durissus terrificus venom.

DETERMINATION OF SURVIVAL TIME OF MICE: The results obtained for the variable survival time of mice are shown in Figure 7. All the tumor-bearing groups treated with Crotalus durissus terrificus venom (G4, G5, G6 and G7), regardless of the dose, presented significantly longer survival time than that of the tumor-bearing group non-treated with venom (G3). In spite of increasing the animals' survival time, the treatment with venom did not cause considerable decrease of mortality.

FIGURE 7.
Distribution of survival time of animals bearing Ehrlich ascites tumor, treated or not with Crotalus durissus terrificus venom. V, venom; EAT, Ehrlich ascites tumor; Statistics: H = 27.54; p < 0.001; G3 < G4, G5, G6 and G7.

DISCUSSION

The analysis of the variable total number of cells present in the peritoneal cavity revealed that all the experimental groups inoculated with tumor cells in the presence or absence of Crotalus durissus terrificus venom, exhibited significantly higher quantities of cells than the saline and venom control groups. This indicates that the increase observed was mainly due to the presence of Ehrlich ascites tumor inoculated in the animals. However, we could observe that the quantity of cells in the experimental groups inoculated with tumor cells and Crotalus durissus terrificus venom was 60% lower than that in the tumor control group. This suggests that in these groups, although the tumor had grown, its growth was slower than that in the tumor control group.

This could also be noticed in the analysis of the variable number of tumor cells. This variable presented a similar behavior to the previous one, i. e., in the tumor-inoculated groups treated with Crotalus durissus terrificus venom, the quantity of tumor cells was about 50% lower than that in the venom control group. These data suggest that Crotalus durissus terrificus venom interfered with the growth of Ehrlich ascites tumor cells during the early phase of treatment, i.e., until the 14 day. Apparently, the treatment induced a slower growth of tumor cells without leading to a considerable elimination of these cells. We could observe that when the treatment with venom was discontinued, the tumor cells presented a normal growth pattern, causing the animals' death in the same rate as in the tumor control group.

A similar fact was reported by Yoshikura et al.(33) who investigated the action of crude venom of Trimeresurus flavoviridis and that of two hemohrragic principles isolated from this venom. These authors conducted in vitro studies using different cell lines and observed that both the crude venom and the hemorrhagic principles caused morphological and physiological changes in tumor cells, without causing cellular death. When these altered cells were removed from the venom-containing culture medium and placed in culture medium without venom, they exhibited normal morphology and physiology and began to grow in the culture medium.

Treatment with Crotalus durissus terrificus did not yield an increase in the number of PMN cells. This could be seen when comparing the venom control group with the saline control group. On the other hand, an increase in the number of PMN cells was observed in the groups inoculated only with tumor cells. According to Freire(13), PMN cells can lead to the elimination of cells that are strange to the host by oxidative and non-oxidative mechanisms. However, in spite of noticing a quantitative increase of PMN cells in the tumor control group, a marked increase in the number of tumor cells was seen. This suggests that PMN cells alone are not capable of inhibiting tumor growth. Conversely, although the quantity of PMN cells was shown to be similar to that of the tumor control group, all the groups inoculated with tumor cells and treated with venom showed an increase in the number of tumor cells that was less significant, i. e., 50% lower than that in the tumor control group.

The number of MN cells was not altered due to the presence of tumor or venom. Most of the MN cells in the peritoneal cavity are macrophages that are the major cells involved in tumor rejection(11,26). However, in spite of the non-occurrence of an increase in the number of MN cells, a significant increase was observed in the activity of peritoneal macrophages in the Crotalus durissus terrificus venom-treated groups. The increase in the macrophage activity was noted mainly in the groups inoculated with larger venom doses. In addition, the group inoculated with the highest venom dose presented the lowest quantity of tumor cells. The increase of macrophage activity, possibly due to the venom, might have been responsible for the slower growth of tumor cells.

It is well known in literature(11,26) that MN cells, mainly macrophages, are the major cells involved in tumor rejection. The variable macrophage spreading revealed that the treatment with Crotalus durissus terrificus venom affects the functional state of macrophages. Figure 6 shows macrophage spreading with increase in size and large cytoplasmic vacuoles. Thus, the antitumor effect observed might be due to an increase of peritoneal macrophage activity and not to an increase in macrophage number.

As already reported by Silva et al.(28), treatment of mice with Crotalus durissus terrificus and Bothrops jararaca venoms brings about an increase of the spreading percentage of peritoneal macrophage compared with the saline control group. It has already been reported in literature that snake venoms can be contaminated by bacteria that are in the snakes' mouth(14,17). This has already been observed for Crotalus durissus terrificus and Bothrops jararaca venoms. The lypopolysaccharide (LPS) present on the bacterial wall is a potent inducer of macrophages and may induce an increase in the percentage of macrophage spreading(1). Therefore, venoms used in scientific research must be dealt with care during the preparation phase to avoid incorrect interpretations.

However, the increase of spreading percentage observed in the present work might not be the result of the presence of bacterial lypopolysaccharide, since the venoms used were carefully centrifuged and filtered before lyophilization to reduce the presence of LPS. However, the presence or absence of LPS in the venom was not tested. The paper by Rabinovitch and DeStefano(22) corroborates the above mentioned assumption. These authors reported(22) that proteolytic enzymes are capable of removing macromolecular components of the cell surface and of affecting certain surface receptors, resulting in a higher affinity of the cell membrane to the substrate. These authors(22) also reported that the treatment of macrophages with tripsin, subtilisin and papain caused dose-dependent increase of percentage of macrophage spreading. Snake venoms are a complex mixture of substances whose characteristics and proportions vary according to the species. Crotalus durissus terrificus venom is composed of amino acids, small peptides, carbohydrates, lipids, biogenic amines and enzymes(4). Thus, as shown by Rabinovitch and DeStefano(22), the increase of spreading percentage observed in this paper after treatment with venom is due to the action of certain enzymes on macrophages.

The analysis of the variable survival time of animals corroborates the idea previously discussed. The treatment with Crotalus durissus terrificus venom produced an increase in the survival time of the animals in all the experimental groups that were inoculated with tumor cells and treated with venom. However, the mortality percentage in these groups was similar to that of the tumor control group. This demonstrates that the presence of venom retards tumor growth.

The animals which were not treated with venom died before those which were treated with venom. When the venom injections were discontinued, the tumor whose growth had been apparently inhibited by the venom, started to grow again and the animals died.

In this study, the results obtained for the evaluation of the antitumor effect of Crotalus durissus terrificus venom are similar to those obtained by Hernandez Plata et al. (15). These authors(15) observed an increase of survival time of the animals. However, crotoxin and crotamin, two fractions of Crotalus durissus terrificus venom, were used by these authors in the treatment, and the tumor line used was rat sarcoma.

On the other hand, the work by Baldi et al.(3) reports results which disagree with those obtained in this work and those by Hernandez Plata et al.(15) Baldi et al.(3) used the same fractions of Crotalus durissus terrificus venom utilized by Hernandez Plata et al.(15) and with several tumor cell lines, among them Ehrlich ascites tumor cells. These authors(3) did not observe any antitumor effect after treatment of Ehrlich ascites tumor with the above mentioned venom fractions. This may be the result of the different treatment procedures used. In this work, we inoculated 1x10 tumor cells in the peritoneal cavity of 30-day-old male mice. The animals received 5 injections of venom in 4 different doses and the treatment started 24 hours after tumor inoculation with a 72-hour interval between injections. Baldi et al.(3) inoculated 60 to 90-day-old female mice with 1x10 tumor cells. The animals were treated with 9 ng of crotoxin and crotamin fractions per gram of the animals' weight, 10 and 18 days after tumor inoculation.

Ehrlich ascites tumor is of rapid growth. With the inoculation of 1x10 tumor cells after 10 days of tumor evolution, mice present about 13x10 cells/ml of liquid extracted after peritoneal washing(11). After 10 days of evolution, with 1x10 tumor cells inoculated in the peritoneal cavity, the quantity of cells would be even higher. Considering that the venom antitumor effect would have a direct effect on tumor cells, the number of cells during treatment might have been very high for the quantity of venom and fractions used. In an attempt to solve this problem, we could increase the venom dose. However, upon increasing the venom dose, another problem would come up, that is, venom toxicity. On the other hand, if the effect of venom were indirect by the stimulation of the host inflammatory response, the number of cells present in the peritoneal cavity might have led to the suppression of the host response due to excess of antigens, preventing the animals from responding to other stimuli such as the venom.

In the present work, the venom doses used were based on the LD50 of each venom. The LD50 for a single injection of Crotalus durissus terrificus venom in the peritoneal cavity of 30-day-old male mice was approximately 0.1 mg/kg. Since the treated animals received 5 venom injections, and since Crotalus durissus terrificus venom is mainly neurotoxic(32), we decided to use doses far below the LD50. However, we observed a higher activity of macrophages in the groups treated with the highest venom dose, i.e., 0.8 and 1.6µg of venom/kg per injection Therefore, if we had used higher doses of Crotalus durissus terrificus venom, respecting the toxicity threshold, we might have obtained better results regarding control of tumor growth.

In addition, the study of fractions of Crotalus durissus terrificus venom associated with different treatment procedures might have revealed a specific fraction of Crotalus durissus terrificus venom that presents a better antitumor activity than that observed for crude venom.

In view of the results obtained in this study, we suggest that according to the experimental design proposed, the inoculation of Crotalus durissus terrificus venom shows a slight action on Ehrlich ascites tumor cells. In a recent review(29), we have already discussed the possible mechanisms involved tumor rejection. Thus, snake venoms might have direct and/or indirect action on tumor cells by stimulating the host cells, mainly macrophages. Such stimulation might induce production and release of several cytokines such as TNF- , IL-1, IL-6 and IL-8. Some of these cytokines have direct cytotoxic effect on tumor cells while others act on other cells and activate them: natural killer cells -NK and cytotoxic T lymphocites. In addition, these cytokines might stimulate production of C-reactive protein and complement factor C3 that would act as opsonins on tumor cells in the liver. The combination of these effects might impede tumor growth and lead to elimination of tumor cells.

In conclusion, for the experimental design proposed, Crotalus durissus terrificus venom was not efficient for complete elimination of tumor cells. In spite of this, the increase in the animals' survival time and the important macrophage stimulation after envenomation are significant results which certainly deserve further studies.

32 VITAL BRAZIL O. Venenos ofídicos neurotóxicos. Rev. Assoc. Med. Bras., 1980, 26, 212-8.

Received 15 August 1996

Accepted 16 September 1996

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CORRESPONDENCE TO:

R. J. DA SILVA - Caixa Postal 577, CEP 18.618-000, Botucatu, São Paulo, Brasil.

Publication Dates

• Publication in this collection
  11 Jan 1999
• Date of issue
  1997

History

• Accepted
  16 Sept 1996
• Received
  15 Aug 1996