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Safety evaluation of the venom from scorpion Rhopalurus junceus: Assessment of oral short term, subchronic toxicity and teratogenic effect
Toxicon 176 (2020) 59–66
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Safety evaluation of the venom from scorpion Rhopalurus junceus: Assessment of oral short term, subchronic toxicity and teratogenic effect Alicia Lagarto a, *, 1, Viviana Bueno a, 1, María R. P�erez b, 2, Caridad C. Rodríguez b, 2, Irania Guevara b, 2, Odalys Vald�es a, 1, Addis Bellma a, 1, Tatiana Gabilondo a, 1, � n a, 1 Alejandro S. Padro a b
Drug Research and Development Center, CIDEM, Ave 26 N 1605 entre Ave Boyeros y Calzada de Puentes Grandes, La Habana, Cuba Laboratories of Biopharmaceuticals and Chemistries Productions, LABIOFAM, Ave Independencia Km 16 1/2 Mulgoba, Boyeros, La Habana, Cuba
A R T I C L E I N F O
A B S T R A C T
Keywords: Rhopalurus junceus Scorpion venom Oral toxicity Teratogenic Toxicological profile
Rhopalurus junceus is the most common scorpion in Cuba and the venom is often used as a natural product for anti-cancer therapy. Despite this, no study has been published concerning its toxicological profile. The aim of the study was characterizing the short-term, subchronic toxicity and the teratogenic potential of Rhopalurus junceus scorpion venom by oral route in mice. Short-term oral toxicity was test in both sexes NMRI mice that received 100 mg/kg/day of scorpion venom extract for 28 days. For the subchronic study, mice were administered with three doses (0.1, 10, and 100 mg/kg) by oral route for 90 days. Teratogenic potential was tested in pregnant mice administered from day 6–15 post conception. Significant differences were observed in body weight and food intake of animal treated for short-term and subchronic assays. Variations in serum urea and cholesterol were observed after 90 days oral treatment. Spontaneous findings not related to the treatment were reveal in histology evaluation. Exposure in pregnant mice did not produce maternal toxicity. Signs of embryo-fetal toxicity were not observed. The current study provides evidence that exposure to low or moderate dose of Rhopalurus junceus scorpion venom by oral route did not affect health of animals and has low impact on reproductive physiology.
1. Introduction
It has previously been reported that soluble venom of this arachnid is not toxic to mice, injected intraperitoneally at doses up to 200 μg/20 g body weight, but it is deadly to insects at doses of 10 μg per animal �mez et al., 2011). The venom causes typical alpha and (García-Go beta-effects on Na þ channels, when assayed using patch-clamp tech niques in neuroblastoma cells in vitro. It also affects Kþ currents con ducted by ERG (ether-a-go-go related gene) channels. The soluble venom was shown to display phospholipase, hyaluronidase and �mez et al., 2011). Furthermore, the anti-microbial activities (García-Go venom from Rhopalurus junceus induces selective and differential anti cancer effect against epithelial cancer cells (Diaz-Garcia et al., 2013) and produced selective cytotoxic effect associated with its apoptosis-inducing effect through the mitochondrial pathway in human breast carcinoma cell line (Diaz-Garcia et al., 2017).
Scorpion venom contains various groups of compounds that exhibit a wide range of biological properties and actions in cells. The general composition and expression level of scorpion venom depends on genetic variation and geographical environment (Song et al., 2012). The scor pion Rhopalurus junceus is an endemic species from Cuba belonging to Buthidae family. This scorpion is widespread and there is no report of scorpionism from this or other species in the country. For this reason, they are not considered dangerous to humans. For a long time, a diluted preparation of venom from Rhopalurus junceus has been used in Cuban traditional medicine for treatment of some illnesses, including cancer, and has shown beneficial effects for some people (Diaz-Garcia et al., 2013; Hern� andez et al., 2009).
Abbreviations: LABIOFAM, Laboratories of Biopharmaceuticals and Chemistries Productions; CENPALAB, Laboratory Animal National Centre; SVE, scorpion venom extract. * Corresponding author. Ave 26 N1605 entre Ave Boyeros y Calzada de Puentes Grandes, Havana City, Cuba. E-mail addresses: [email protected] (A. Lagarto), [email protected] (V. Bueno). 1 [email protected] 2 [email protected] https://doi.org/10.1016/j.toxicon.2020.02.002 Received 21 October 2019; Received in revised form 28 January 2020; Accepted 3 February 2020 Available online 11 February 2020 0041-0101/© 2020 Elsevier Ltd. All rights reserved.
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The use of Rhopalurus junceus scorpion venom in humans needs a safety evaluation for oral route of administration. Available data are insufficient to support the safety of this preparation by oral route. The overall objective of this investigation was to characterize the short-term, subchronic toxicity and the teratogenic potential of Rhopalurus junceus scorpion venom by oral route in mice. An additional aim was to identify the no-observed-adverse-effect levels (NOAELs) for subchronic dosing and exposure during organogenesis period.
Spectronic Genesys 2. 2.4. Subchronic toxicity test Mice were randomized to four groups of each sex (4–5 weeks of age, 18–20 g of body weight, n ¼ 10 per group). Animals were exposed to SVE by gavage for 90 days as described above. The dosages delivered were 0.1, 10 and 100 mg/kg/day. Animals of control groups did not received treatment. Animals were monitored for body weight, food consumption, and behavioral and clinical signs as described in the shortterm toxicity study. At the completion of the subchronic study, blood samples were collected as described above and serum obtained for he matological and biochemical analyses (OECD, 1998). After collection of blood samples, mice were euthanized by exsan guination. Selected organs for weight were liver, kidneys, adrenals, spleen, thymus, brain, heart, ovaries, epididimos and testes. Selected organs (heart, kidneys, liver, spleen, brain, lungs, esophagus, stomach, intestines, thymus, adrenals, thyroid, parathyroid, trachea, pancreas, femur, bone marrow, salivary glands, cervical ganglion, epididimos, gonads, prostate, uteru, ovaries, seminal bladder and femoral muscle) were removed, fixed, sectioned, and stained for histopathological ex amination (OECD, 1998). A satellite group of 10 animals per sex (5 control and 5 treated) was used in the top dose group for observation, after the treatment period, for reversibility or persistence of any toxic effects. In these groups blood and organs were taken 28 days after the end of the 90 day treatment period. Additionally, hematological and clinical biochemistry analysis were made with animals before dosing to establish the range of control value (OECD, 1998).
2. Materials and methods 2.1. Test substances Adults Rhopalurus junceus scorpions were maintained in individual plastic cages in laboratories belonging to Laboratories of Bio pharmaceuticals and Chemistries Productions (LABIOFAM). Venom from scorpions kept alive in the laboratory was extracted by electrical stimulation. Venom was dissolved in distilled water and centrifuged at 15000�g for 15min. The supernatant was filtered by using a 0.2 μm syringe filter and stored at 20 � C until used. The protein concentration was calculated by the Lowry modified method. A solution with a protein concentration of 8–11 mg/ml was used in the studies. 2.2. Animals Animal care was performed in conformity with the Guide for the Care and Use of Laboratory Animals (National-Research-Council and Press, 2011). Healthy both sexes NMRI albino mice weighing 22 � 2 g were used in the studies. Animals were obtained from the Laboratory Animal National Centre (CENPALAB), Havana, Cuba and were randomly assigned to dosage groups. Animals was were housed together by sex in polycarbonate cages in a light- and humidity-controlled biohazard suite (24 � 2 � C; 55 � 5% relative humidity), with a 12-h light-dark cycle, and free access to drinking water and a standard laboratory diet CMO1000 (CENPALAB). Experiments were conducted in accordance with UE Directive 2010/63/EU for animal experiments (EUROPEAN-PARLIA MENT, 2010). The experimental protocols were approved by the Insti tutional Ethical Committee.
2.5. Evaluation of teratogenic effect Mature and healthy NMRI mice (female of 25 � 2 g and male of 30 � 2 g) were housed in plastic cages and quarantined for one week before mating. Four females were cohabited overnight with a male and examined for the presence of vaginal plug in their vagina. The day that vaginal plug was detected was defined as gestational day 0. Pregnant mice were randomized in control and treated groups (20 animals per group) and administered by intragastric intubation (gavage) at doses of 0.1, 10 and 100 mg/kg/day on gestational days 6–15 (ICH, 2005). Pregnant mice were observed daily for clinical signs and mortality. Body weight and food intake were monitored three times a week. Dams were killed by diethyl ether anesthesia and necropsy was performed on gestational day 18. Blood was taken from orbital sinus and examined for hematology (hemoglobin, hematocrit, erythrocyte and leukocyte count) and biochemical parameters (glucose, total cholesterol, aspartate aminotransferase (AST), and alkaline phosphatase). Macroscopic ex amination of organs was conducted for all dams, and organs with macroscopic damage were preserved for histological evaluation. Dams and fetus were examined as described previously (Burdan et al., 2005; Claudio et al., 1999; Lagarto et al., 2013).
2.3. Short-term toxicity test The study was designed to monitor the effect of a top dose on clinical signs, body weight, biochemical and hematological parameters previous to 90 days toxicity study. Mice were randomized to two groups of each sex (5–6 weeks of age, 24–27 g of body weight, n ¼ 5 per group). Ani mals were exposed to 100 mg/kg of scorpion venom extract (SVE) by gavage for a 28-day period. The dose selected was the top dose possible according to the nature of the substance and was ten times the thera peutic dose in preclinical studies. Administration volume was adjusted every 7 days according to body weight change. Another 5 mice of each sex were assessed for control without treatment. Animals were moni tored weekly for body weight and food consumption. Both behavior and clinical signs were monitored daily. Blood samples were collected in fasted overnight animals under anesthesia of Tiopental (40 mg/kg) on day 28 of dosing for hematology and clinical biochemistry. Gross pa thology was performed to all animal for organs and tissues (OECD, 2008). Hematology included determination of hematocrit by micro hematocrit capillaries, hemoglobin concentration by diagnostic kit produce by Biologic Products Inc. Havana, Cuba, erythrocyte and leukocyte count in Neubauer chamber, and differential leukocyte count by extension in microscopy slides. Clinical biochemistry included glucose, total cholesterol, creatinine, urea, albumin, alanine amino transferase (ALT) and alkaline phosphatase. The measures were made with diagnostic kit produced by Biologic Products Inc. Havana, Cuba. The absorbance values were determined in a spectrophotometer
2.6. Statistics Results were expressed as the mean � SEM. All statistical analysis was assessed using the GraphPad Prism Version 7 (GraphPad Software, San Diego, California, USA). Each test group was compared with control. Bartlett’s test was applied to determine whether the variance was ho mogeneous or not. When the variance was homogenous, one-way ANOVA was applied. When the variance was not homogenous, the Kruskal–Wallis test (nonparametric ANOVA) was performed. Statistical differences in the means among the groups were analyzed by means of Dunnett’s multiple comparison test. The parameters of developmental toxicity in fetuses were expressed with the litter as basic unit. Incidences of visceral and skeletal malformations/variations were analyzed by means of Fisher exact probability. A Two-sided analysis with a p-value of 60
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in urea and cholesterol levels in animals treated with SVE was reversible (Table 2). Similar histological findings were observed in treated and control animals.
0.05 or 0.01 was performed to determine statistical significance. 3. Results 3.1. Short-term toxicity test
3.3. Evaluation of teratogenic effect
Signs of toxicity were not observed during the experimental period in treated groups. Body weight trend were affected in treated groups. In male treated animals, body weight was significantly lower than control group throughout the study. In female, significant diference was observed until the second week of the experiment. Significantly lower body weight gain was observed in both sexes treated animal. In female treated animals, food consumption was significantly lower than control group (Fig. 1). No statistical differences between treated and control groups were observed in biochemical and hematological parameters.
Maternal toxicity: No evident clinical signs were observed in any dam during the study. There were no differences between the treated groups and controls for the maternal weight gain, maternal body weight in crease during treatment, gravid uterine weight, maternal net weight gain, food intake, or hematology and biochemical parameters (Table 3). Macroscopic finding of organs and tissues were not observed in dams. Embryo-fetal Toxicity: Statistical difference was not observed in number of implantation sites, corpora lutea, live and dead fetuses, as well as for resorptions, pre-implantation and post-implantation loss between SVE-exposed groups and the control (Table 4). Types and fre quencies of external abnormalities observed in fetuses are shown in Table 5. Changes affected paws and head. No statistical difference was observed in frequency of external variations and malformations compared to control group. Skeletal and visceral abnormalities were evaluated in high dose exposed fetuses (Table 6). The visceral malformations and variations observed in the present study were limited to the brain at low incidence. Three litters in control and one litter in high dose group miss for skeletal examination due to damage during the fixation process. All skeletal changes were limited to the axial skeleton and lumbar arch. These findings were spontaneous and unrelated to the drug. Oral treatment with SVE at 100 mg/kg on gestational day 6–15 neither induced skeletal abnormalities nor affected visceral morphology.
3.2. Subchronic toxicity test Signs of toxicity and mortality were not observed during the 90 day experimental period in treated groups. A significant variation in body weight trends was observed during the study period in both sexes (Fig. 2). In addition, food intake was significant affected in all doses in male. Low and high dose treated groups were the most affected. Food intake in medium dose group was significant decreased only at 6th and 7th weeks. In female, food intake was less affected than male. Significant differences were observed in low and medium dose groups only in a few weeks. Body weight gain (three doses) was significant affected in male. Dose effect relationship was assed for body weight gain by lineal regresion being significant for female (Fig. 3). No treatment-related changes were observed in hematological pa rameters tested during the study period. Clinical chemistry examination showed a decrease in urea level at 10 mg/kg/day (male) and 100 mg/ kg/day (both sexes). Additionally, cholesterol level was decrease after 90 days of oral treatment in male being significant at 100 mg/kg/day. The others biochemical parameters were not affected by dosing with SVE (Table 1). Any statistical variation was not observed in the relative organ weight (as % of total body weight) compared to control group. There was no treatment related histological changes in animals treated with high dose and control group in both sexes. Mandibular lymph node lymphoma, liver inflammation (hepatitis) and pneumonia were observed in one male of control group. In female control group, one animal with bronchitis and tracheitis was observed. In male treated group, pneumonia was observed in one animal. In female treated group, tracheitis, pneumonia, focal pancreatitis and liver microgranuloma were observed in one animal (Fig. 4). Satellite Group Results. Variations observed in body weight, weight gain and food intake in male treated animals remained significantly lower than controls 28-days after treatment (Fig. 5). Decrease observed
4. Discussion Scorpion and its products have been used as a traditional Chinese medicine for thousands of years. It has previously been reported that crude scorpion venom or isolated peptides from scorpion venom may inhibit the proliferation of cancer cells and induce cell apoptosis (Ding et al., 2014; Song et al., 2012). Scorpion venom contains peptides which exhibit anti-microbial properties. Kievit et al. reported that an ingre dient in the venom of the “death stalker” scorpion could help in gene therapy for effective treatment for brain cancer (Kievit et al., 2010). Rhopalurus junceus scorpion is the most common scorpion in Cuba. The venom showed cytotoxic effect in murine myeloma and rat prostate tumor cell lines (Hern� andez et al., 2009) and induces selective and differential anticancer effect against cancer cells (Diaz-Garcia et al., 2017, 2013). Enzymatic analysis of venom showed low enzymatic ac tivity, which could contribute to the low toxic potential of this scorpion venom (Diaz-Garcia et al., 2015). However, there was insufficient literature available of safety evaluation of products from scorpion venom and recent researches mainly focus on pharmacology evaluation and other relevant topics but none of them put emphasis on safety
Fig. 1. Body weight trends and gains and food intake during short-term toxicity test. The data are expressed as mean � SEM of 5 animals, *p < 0.05 **p < 0.01 ***p < 0.001 (significantly different from control). 61
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Fig. 2. Body weight trends and food intake during subchronic toxicity test in male (A–B) and female (C–D). The data are expressed as mean � SEM of 10 animals, *p < 0.05 **p < 0.01 ***p < 0.001 (significantly different from control).
Fig. 3. Body weight gain during subchronic toxicity test (A) and doses effect relationship (B). The data are expressed as mean � SEM of 10 animals, *p < 0.05 **p < 0.01 (significantly different from control).
evaluation. For safety evaluation, SVE was orally given at doses up to 100 mg/ kg/day to male and female mice. Our results indicate that oral exposure to SVE affect weight trends. In fact, a significant difference in weight gain was observed in male. Previous study indicate that the intravenous injection of toxin-γ from Tityus serrulatus scorpion venom induces a rapid, intense and sustained inhibition of gastric emptying (Bucaretchi et al., 1999). Sofer et al. reported that Leiurus quinquestriatus scorpion venom provoked gastrointestinal ischemia in pigs (Sofer et al., 1997) whereas Gwee et al. and Teixeira et al. respectively, demonstrated that L. quinquestriatus and T. serrulatus scorpion venoms were capable of relaxing rat isolated anococcygeus muscle and rabbit isolated corpus cavernosum via the local release of nitric oxide (Gwee et al., 1995; Teixeira et al., 1998). Pharmacokinetics and biodistribution of SVE in mice showed 30% of bioavailability by oral route and stomach, kidney, spleen and lung showed the major uptakes for 131I-labelel venom (Dia z-Garcia et al., 2019). These observations are important since a decrease
in gastrointestinal blood flow and the local release of nitric oxide may inhibit gastric smooth muscle cell activity (Chou, 1982; Shah et al., 2004). Thus, the effect on weight trends observed could be due to in hibition of gastric emptying by some components of SVE causing en hances satiety and decrease the food intake. Female mice were less affected than male. Female did not present significant alterations in weight gain and food intake was minimal affected by treatment. How ever, a dose effect ratio was observed in weight gain. Variations in body weight and food intake did not affect the integrity of organs and tissues and did not influence the health condition of the animals. Hematological parameters were not significant affected by sub chronic dosing with SVE. Significant decrease in serum urea was observed in both sexes treated animals. Common causes of decrease in urea concentration includes severe liver disease, low protein diet, severe polyuria/polydipsia and drugs. Absent of variations in liver enzymes and histopathological lesions excludes liver disease factor. On the other hand, variations in food intake could be associated with reduced urea 62
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production but the same dose groups for food intake and variations in urea concentration were not affected. In male animals, food consump tion was more affected in low and high dose groups than medium dose group. Serum urea variations were significant in medium and high dose groups. In female animals, were more evident that effects on food intake and urea are not related. However, variations in urea concentration could be related to the SVE oral exposure. High dose groups treated animals of both sexes and medium dose group in male animals were significant affected. SVE oral exposure up to 28 days did not affect urea concentrations at high dose and results of satellite groups in oral 90 days study showed that the effect on urea was reversible. The decrease in urea may be related to low protein absorption in the gastrointestinal tract or polyuria/polydipsia but further studies are required to clarify the causes. On the other hand, there is no evidence that the effect on urea concentration represent any functional impairment in treated animals and there is no related to a change in other parameters. The results are close to control range and the severity of the change is minimal. For that, we consider that the urea variation observed in treated animals does not constitute an adverse effect. Not significant decrease in creatinine concentration was observed in male treated animals. These results are within normal biological varia tion. Values are within control range used in the study and within reference values for this strain (Aleman et al., 2000). Variation in cholesterol observed was minimal and close to control range. This change did not affect health state of the animals and was reversible. For that, these variations are considered without pathological significance. Findings in histopathological exam were minimal and include very common lesions reported in mice. Tracheitis is a frequent finding of small foci of inflammatory cell infiltrates with low incidence, sporadic finding resulting from infections caused by gavage accidents. Bronchitis and pneumonia are especially common in females with small foci of inflammatory cells, predominantly lymphocytes. Pancreatitis as a small aggregates of lymphoid cells are common in mice. Liver inflammation (hepatitis) is a very common finding, up to 50% of CD-1 mice have small foci of both acute and chronic inflammatory cells scattered in the liver
Table 1 Results of biochemical parameters following 90-days exposure to SVE. Values represent the mean � SEM (n ¼ 10), **p < 0.01 significantly different from control. Treatment
Control
Scorpion venom extract
Doses (mg/kg/ day) Male Alkaline phosphatase (U/l) ALT (U/l)
0
0.1
10
100
146.1 � 15.0
125.7 � 11.1
137.1 � 12.7
118.6 � 8.9
109–183
Creatinine (μmol/l) Glucose (mmol/l)
63.9 � 6.4 4.4 � 0.5
Cholesterol (mmol/l) Albumin (g/l)
3.2 � 0.2 14.2 � 1.3
36.8 � 7.3 6.5 � 0.7 61.7 � 5.5 4.3 � 0.7 2.7 � 0.3 12.8 � 1.1
39.5 � 11.5 5.2 � 0.4** 46.0 � 3.4 4.9 � 0.2 2.5 � 0.2 13.8 � 0.7
23.5 � 6.1 5.4 � 0.7** 45.8 � 7.4 3.8 � 0.4 2.2 � 0.1** 14.5 � 0.8
13.6–42.4
Urea (mmol/l)
28.5 � 5.9 8.4 � 0.5
149.3 � 13.2
137.9 � 11.0
165.0 � 23.5
150.8 � 13.7
117–182
Creatinine (μmol/l) Glucose (mmol/l)
55.2 � 10.3 4.9 � 0.5
Cholesterol (mmol/l) Albumin (g/l)
1.9 � 0.4
24.5 � 5.5 5.1 � 0.5 44.2 � 5.9 4.4 � 0.9 1.2 � 0.1 12.6 � 0.6
26.0 � 6.0 6.2 � 0.7 47.4 � 5.2 4.6 � 0.3 1.9 � 0.1 14.8 � 0.6
18.2 � 3.8 3.3 � 0.2** 60.0 � 11.5 4.8 � 0.5 1.9 � 0.1 14.7 � 0.3
15.2–35.8
Urea (mmol/l)
25.5 � 4.2 6.2 � 1.0
Female Alkaline phosphatase (U/l) ALT (U/l)
13.0 � 0.8
Control Range
6.9–9.9 38.7–69.0 3.1–5.7 2.4–3.9 10.9–17.4
4.3–9.4 28.8–81.7 3.8–6.0 1.0–2.8 10.6–15.3
Fig. 4. Photomicrograph showing histological findings in representative animal. (A) Control lung with normal histology. (B) Pneumonia observed in female treated animals. (C) Control liver with normal histology. (D) Inflammation (hepatitis) with necrosis observed in one male control animal (hematoxylin and eosin stain). 63
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Fig. 5. Body weight trends and food intake during subchronic toxicity test in satellite group. The data are expressed as mean � SEM of 5 animals, *p < 0.05 **p < 0.01 ***p < 0.001 (significantly different from control). Table 2 Biochemical results of animals 28 days after 90-days exposure to SVE. Values represent the mean � SEM (n ¼ 5). Parameter
Urea (mmol/l) Cholesterol (mmol/ l)
Male
Table 3 Effect of SVE on maternal toxicity end points in mice. Values represent the mean � SEM (n ¼ 20).
Female
Control
100 mg/kg/ day
Control
100 mg/kg/ day
9.8 � 1.8 3.3 � 0.8
8.7 � 0.9 2.8 � 0.7
10.4 � 1.7 2.6 � 0.4
9.7 � 0.5 3.1 � 0.6
Treatment
Control
Scorpion venom extract
Dosage (mg/kg) No. of pregnant animals treated. No. of animals at term gestation with live fetuses. Initial body weight (GD 0/g�SEM).
0 20 20
0.1 21 20
10 20 20
100 20 20
26.2 � 0.5 2.3 � 0.4
27.0 � 0.5 2.1 � 0.3 12.5 � 0.4 9.2 � 0.6 23.9 � 0.8 16.9 � 0.5 7.0 � 0.6 5.4 � 0.2
26.1 � 0.4 2.4 � 0.4 12.9 � 0.5 9.0 � 0.6 24.4 � 0.8 16.5 � 0.8 7.5 � 0.5 5.6 � 0.1
25.6 � 0.4 1.8 � 0.3 12.5 � 0.4 8.4 � 0.6 22.7 � 0.8 16.1 � 0.6 6.6 � 0.5 5.4 � 0.2
11.1 � 0.8 39.1 � 1.5 5.57 � 0.6 4.52 � 0.6
10.7 � 0.6 40.0 � 1.1 5.70 � 0.5 5.75 � 0.7
9.5 � 0.8 39.8 � 1.3 5.59 � 0.4 5.04 � 0.7
9.4 � 0.9 39.7 � 1.6 5.76 � 0.4 3.76 � 0.3
4.2 � 0.4
3.9 � 0.2 3.6 � 0.3 73.0 � 3.6 85.9 � 4.2
4.1 � 0.3 3.4 � 0.4 77.1 � 4.3 84.8 � 4.5
3.9 � 0.3 2.7 � 0.3 76.9 � 4.7 91.9 � 5.4
Maternal body weight gain (GD 0–6/g�SEM). Maternal body weight gain (GD 6–15/g�SEM). Maternal body weight gain (GD 15–18/g� SEM). Maternal body weight gain (GD 0–18/g� SEM). Gravid uterine weight at term (g�SEM). Maternal net weight gain (g� SEM). Food intake (g/animal/day� SEM)
(Peckham, 2002). Considering the incidence and nature of the histo logical changes they are spontaneous and unrelated to the treatment. In the teratological study conducted in a single rodent species, adverse reproductive effects were not observed after the administration of three doses of SVE by oral gavage. Maternal toxicity was not observed because of exposure to the test substance by clinical observations, body weight gain, food intake, hematology and biochemical parameters and necropsy finding. Slight increase of pre-implantation loss was observed with 10 mg/kg treated animals. This change was without statistically significant and not related to the treatment because the SVE exposure began on day 6 post conception and the variations occurred before im plantation. External anomalies were similar for all experimental groups. Incidence of hemorrhage does not represent an anomaly and it is considered because of a functional disorder and thus not strictly devel opmental anomaly. For that reason, hemorrhage was classified as “Not Malformation” (Solecki et al., 2003). The total number of litters and fetuses with visceral and skeletal malformations and variations were similar in all experimental groups. We considered that these finding was spontaneous and unrelated to the drug. There are not studies in literature about the toxic effect of the venom from the scorpion Rhopalurus junceus administered by oral route in ro dents. It is known that the scorpion venom can cause malformations or behavioral defects to the offspring of mothers exposed to the venom during pregnancy. Previous reports reflected the effect of prenatal exposure to Tytus bahiensis scorpion venom. A moderate dose of this venom injected to pregnant rats was able to elicit alterations in physical and behavioral development in the offspring during the postnatal period
Hematology Hemoglobin (mmol/L� SEM) Hematocrit (%�SEM) Total erythrocyte count (cell/ mm3x106�SEM) Total leukocyte count (cell/mm3 x103�SEM) Biochemistry Glucose (mmol/L�SEM)
24.9 � 0.8 17.4 � 0.6 7.8 � 0.5 5.5 � 0.2
Total cholesterol (mmol/L�SEM)
3.5 � 0.3
AST (U/L�SEM)
78.5 � 6.2 93.4 � 4.1
Alkaline phosphatase (U/L�SEM)
64
13.7 � 0.6 9.0 � 0.5
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Table 4 Effect of SVE on embryo-fetal parameters in mice. Values represent the mean � SEM (n ¼ 20). Treatment
Control
Scorpion venom extract
Dosage (mg/kg) Litters with live fetuses. No. of corpora lutea (Mean � SEM)/litter. Mean � SEM of implantations/litter No. of implantations. No. of live fetuses (Mean � SEM)/litter. No. of dead fetuses (litters affect) No. of early resorptions (litters affect) No. of late resorptions (litters affect) Pre-implantations loss (%) Post-implantation loss (%) Mean placenta weight (g � SEM)/litter. Mean fetal body weight (g � SEM)/litter. 1. Male
0 20 12.8 � 0.4 12.6 � 0.4 251 11.6 � 0.5 1(1)
0.1 20 12.6 � 0.4 12.3 � 0.4 246 11.2 � 0.4 1(1)
10 20 12.6 � 0.4 11.5 � 0.6 230 10.8 � 0.5 1(1)
100 20 11.6 � 0.5 11.4 � 0.4 227 10.6 � 0.5 1(1)
11(10)
13(9)
7(6)
10(7)
6(3)
9(8)
5(4)
4(3)
1.95 7.17 0.14 � 0.01 1.29 � 0.03 1.31 � 0.03 1.24 � 0.05 3.0 � 0.5
2.77 9.35 0.13 � 0.006 1.30 � 0.02 1.30 � 0.02 1.28 � 0.02 3.0 � 0.5
8.73 6.09 0.13 � 0.006 1.33 � 0.02 1.32 � 0.02 1.34 � 0.03 3.5 � 0.5
1.73 7.05 0.13 � 0.008 1.31 � 0.03 1.32 � 0.03 1.29 � 0.03 2.9 � 0.4
2. Female Sex ratio (M/F).
Table 6 Visceral and skeletal defects following oral maternal exposure to SVE. Values represent the mean � SEM (n ¼ 20).
Treatment
Control
Scorpion venom extract 0.1 223 (20)
10 216 (20)
100 211 (20)
3(3) 1(1) 1(1) 0 1(1) 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0
0 0
0 0
2(2)
4(4)
3(3)
Control
Scorpion venom extract
Dosage (mg/kg) Number fetuses (litters) examined – Visceral Exam Visceral malformations No. of fetuses with malformations Cerebrum – Misshapen Visceral variations No. of fetuses with variations Brain - Hemorrhage Lateral cerebral ventricle – Dilated Number fetuses (litters) examined – Skeletal Exam Skeletal malformations No. of fetuses with malformations Lumbar arch - Misshapen Sternebra - Absent Xiphisternum - Absent Skeletal variations No. of fetuses with variations Rib - Misaligned Xiphisternum – Bipartite ossification Xiphisternum- Incomplete ossification Sternebra- Incomplete ossification Sternebra - Asymmetric
0 100 118 104(20) (20) Fetuses affected (litters affected) 0 1(1) 0 1(1) 3(3) 6(2) 3(3) 1(1) 0 5(1) 96(17) 101(19) 1(1)
4(3)
1(1) 0 0 2(2) 2(2) 0 0 0 0
1(1) 2(2) 1(1) 9(5) 2(2) 2(1) 2(2) 4(2) 1(1)
Published preliminary online data refer antitumoral activity in human (Grupo M� edico LifEscozul, 2018). Oral doses referred in traditional medicine are usually low, the use of the diluted preparation (6 ml four times a day) in patients with anal and rectal cancer after surgery has been reported (Poch Mulgado et al., 1999). Despite the traditional use, Rhopalurus junceus scorpion venom should be used with caution for the lack of safety and effective evaluation by oral route. In summary, 100 mg/kg/day orally was the subchronic NOAEL for Rhopalurus junceus scorpion venom, for effects other than variations in weight gain that did not affect the health state of the animals. The oral exposure in mice up to 100 mg/kg/day during organogenesis period did not induce significant maternal and embryo-fetal toxicity. The current study provides evidence that exposure to low or moderate dose of SVE by oral route did not affect health of animals and has low impact on reproductive physiology. Nevertheless, additional studies are necessary for a complete risk assessment and traditional use should be done with caution.
Table 5 External malformations and variations following oral maternal exposure to SVE. Values represent the mean � SEM (n ¼ 20). Dosage (mg/kg) 0 Number fetuses (number litters) 233 examined (20) External malformations No. of fetuses with malformations 8(2) Meningo-encephalocele 0 Exencephaly 1(1) Tongue - Absent 2(1) Hindlimb – Malrotated 0 Palate - Cleft 5(1) External variations No. of fetuses with variations 1(1) Palatal rugae - Absent 1(1) External not malformation (unclassified) Hemorrhage 5(4)
Treatment
CRediT authorship contribution statement Alicia Lagarto: Conceptualization, Methodology, Validation, Formal analysis, Writing - original draft, Writing - review & editing, Project administration. Viviana Bueno: Investigation, Data curation. �rez: Conceptualization, Methodology, Investigation, María R. Pe Writing - review & editing. Caridad C. Rodríguez: Conceptualization, Methodology, Investigation, Writing - review & editing. Irania Gue vara: Conceptualization, Methodology, Investigation, Writing - review �s: Investigation, Data curation. Addis Bellma: & editing. Odalys Valde Investigation, Data curation. Tatiana Gabilondo: Investigation, Data � n: Investigation, Supervision. curation. Alejandro S. Padro
(Barao et al., 2008; Dorce et al., 2009), while low subcutaneous doses of Tytus bahiensis venom did not cause maternal or clear fetal toxicity (Cruttenden et al., 2008). Rhopalurus junceus is known by the vulgar name of “Blue Scorpion” and a diluted solution of its venom known as “Escozul” (a trade mark) is an alternative drug used for pain relief, anti-inflammatory and cancer �mez et al., 2011; Yglesias-Rivera treatment, mainly in Cuba (García-Go and Díaz-García, 2018). There are several communications dealing with anticancer in vitro effect of the venom against cancer cell lines from epithelial origin without affecting normal cells, in the cervical cancer cell line HeLa (Diaz-Garcia et al., 2013; Yglesias-Rivera et al., 2019) and the metastatic breast cancer cell line, MDA- MB-231 (Diaz-Garcia et al., 2017). There are few effective assays performed in vivo, none orally. Significant tumor growth delay in the murine mammary adenocarci noma F3II and dose-dependent decrease of Ki-67 in tumor-bearing ani mals treated intraperitoneally at 0.2; 0.8; 3.2 mg/kg doses were reported recently (Díaz-García et al., 2019). However, oral treatment with Escozul in Cuban traditional medicine for cancer treatment has shown beneficial effects for some people.
References Aleman, C.L., Noa, M., Mas, R., Rodeiro, I., Mesa, R., Menendez, R., Gamez, R., Hernandez, C., 2000. Reference data for the principal physiological indicators in three species of laboratory animals. Lab. Anim. 34, 379–385. https://doi.org/ 10.1258/002367700780387741. Barao, A.A., Bellot, R.G., Dorce, V.A., 2008. Developmental effects of Tityus serrulatus scorpion venom on the rat offspring. Brain Res. Bull. 76, 499–504. https://doi.org/ 10.1016/j.brainresbull.2008.02.033. Bucaretchi, F., Vinagre, A.M., Chavez-Olortegui, C., Collares, E.F., 1999. Effect of toxin-g from Tityus serrulatus scorpion venom on gastric emptying in rats. Braz. J. Med. Biol. Res. 32, 431–434. https://doi.org/10.1590/s0100-879x1999000400009.
65
A. Lagarto et al.
Toxicon 176 (2020) 59–66 Hern� andez, O., Casado, I., et al., 2009. Evaluaci� on de la toxicidad in vitro del veneno del alacr� an Rophalurus junceus a trav�es de un ensayo celular. Rev. Cubana Invest. Biom�ed. 28. ICH, 2005. Detection of Toxicity to Reproduction for Medicinal Products & Toxicity to Male Fertility. S5(R2). Kievit, F.M., Veiseh, O., Fang, C., Bhattarai, N., Lee, D., Ellenbogen, R.G., Zhang, M., 2010. Chlorotoxin labeled magnetic nanovectors for targeted gene delivery to glioma. ACS Nano 4, 4587–4594. https://doi.org/10.1021/nn1008512. Lagarto, A., Bellma, A., Couret, M., Gabilondo, T., L� opez, R., Bueno, V., Guerra, I., Rodríguez, J., 2013. Prenatal effects of natural calcium supplement on Wistar rats during organogenesis period of pregnancy. Exp. Toxicol. Pathol. 65, 49–53. https:// doi.org/10.1016/j.etp.2011.05.009. National-Research-Council, Press, N.A., 2011. Guide for the Care and Use of Laboratory Animals, eighth ed. (Washington DC). OECD, 2008. Test No. 407: Repeated Dose 28-day Oral Toxicity Study in Rodents, OECD Guidelines for the Testing of Chemicals. OECD Publishing, Paris. https://doi.org/ 10.1787/9789264070684-en. OECD, 1998. Test No. 408: Repeated Dose 90-Day Oral Toxicity Study in Rodents, OECD TG 408. Guideline for the Testing of Chemicals. Peckham, J.C., 2002. Animal histopathology. In: Derelanko, M.J., Hollinger, M.A. (Eds.), Handbook of Toxicology. CRC Press, pp. 668–692. Poch Mulgado, E., Formental Planas, J.J., Alvarez Lambert, K.R., Bordier Chibas, M., Frometa Gomez, A.A., Andalia Ricardo, E., 1999. Uso Escozul ( Toxina De Alacran ) En Pacientes Operados Cancer. Anal. Guantanamo. Shah, V., Lyford, G., Gores, G., Farrugia, G., 2004. Nitric oxide in gastrointestinal health and disease. Gastroenterology 126, 903–913. https://doi.org/10.1053/j. gastro.2003.11.046. Sofer, S., Cohen, R., Shapir, Y., Chen, L., Colon, A., Scharf, S.M., 1997. Scorpion venom leads to gastrointestinal ischemia despite increased oxygen delivery in pigs. Crit. Care Med. 25, 834–840. https://doi.org/10.1097/00003246-199705000-00020. Solecki, R., Bergmann, B., Burgin, H., Buschmann, J., Clark, R., Druga, A., Van Duijnhoven, E.A., Duverger, M., Edwards, J., Freudenberger, H., Guittin, P., Hakaite, P., Heinrich-Hirsch, B., Hellwig, J., Hofmann, T., Hubel, U., Khalil, S., Klaus, A., Kudicke, S., Lingk, W., Meredith, T., Moxon, M., Muller, S., Paul, M., Paumgartten, F., Rohrdanz, E., Pfeil, R., Rauch-Ernst, M., Seed, J., Spezia, F., Vickers, C., Woelffel, B., Chahoud, I., 2003. Harmonization of rat fetal external and visceral terminology and classification. Report of the fourth workshop on the terminology in developmental toxicology, berlin, 18-20 April 2002 Reprod. Toxicol. 17, 625–637. Song, X., Zhang, G., Sun, A., Guo, J., Tian, Z., Wang, H., Liu, Y., 2012. Scorpion venom component III inhibits cell proliferation by modulating NF-kappaB activation in human leukemia cells. Exp. Ther. Med. 4, 146–150. https://doi.org/10.3892/ etm.2012.548. Teixeira, C.E., Bento, A.C., Lopes-Martins, R.A., Teixeira, S.A., von Eickestedt, V., Muscara, M.N., Arantes, E.C., Giglio, J.R., Antunes, E., de Nucci, G., 1998. Effect of Tityus serrulatus scorpion venom on the rabbit isolated corpus cavernosum and the involvement of NANC nitrergic nerve fibres. Br. J. Pharmacol. 123, 435–442. https://doi.org/10.1038/sj.bjp.0701623. Yglesias-Rivera, A., Díaz-García, A., 2018. Effect of Rhopalurus junceus scorpion venom on inflammation-related cytokines in healthy BALB/C mice. Ann. Microbiol. Immunol. 1, 1007. Yglesias-Rivera, A., Rodríguez-S� anchez, H., Díaz-García, A., 2019. Synergistic effect of Rhopalurus junceus scorpion venom combined with conventional cytostatics in cervical cancer cell line HeLa. J. Pharm. Pharmacogn. Res. 7, 67–76.
Burdan, F., Szumilo, J., Dudka, J., Klepacz, R., Blaszczak, M., Solecki, M., Korobowicz, A., Chalas, A., Klepacki, J., Palczak, M., Zuchnik-Wrona, A., HadalaKis, A., Urbanowicz, Z., Wojtowicz, Z., 2005. Morphological studies in modern teratological investigations. Folia Morphol. (Warsz) 64, 1–8. Chou, C.C., 1982. Relationship between intestinal blood flow and motility. Annu. Rev. Physiol. 44, 29–42. https://doi.org/10.1146/annurev.ph.44.030182.000333. Claudio, L., Bearer, C.F., Wallinga, D., 1999. Assessment of the U.S. Environmental Protection Agency methods for identification of hazards to developing organisms, Part II: the developmental toxicity testing guideline. Am. J. Ind. Med. 35, 554–563. https://doi.org/10.1002/(sici)1097-0274(199906)35:6<554::aid-ajim2>3.0.co;2-x. Cruttenden, K., Nencioni, A.L., Bernardi, M.M., Dorce, V.A., 2008. Reproductive toxic effects of Tityus serrulatus scorpion venom in rats. Reprod. Toxicol. 25, 497–503. https://doi.org/10.1016/j.reprotox.2008.04.011. Diaz-Garcia, A., Morier-Diaz, L., Frion-Herrera, Y., Rodriguez-Sanchez, H., CaballeroLorenzo, Y., Mendoza-Llanes, D., Riquenes-Garlobo, Y., Fraga-Castro, J.A., 2013. In vitro anticancer effect of venom from Cuban scorpion Rhopalurus junceus against a panel of human cancer cell lines. J. Venom Res. 4, 5–12. Diaz-Garcia, A., Pimentel Gonzalez, G., Basaco Bernabeu, T., Rodriguez Aurrecochea, J. C., Rodriguez Sanchez, H., Sanchez Monzon, I., Hernandez Gomez, M., Rodriguez Torres, C., Regla Rodriguez Capote, M., Guevara Orellanes, I., 2019. Pharmacokinetics and biodistribution of Rhopalurus junceus scorpion venom in tumor-bearing mice after intravenous and oral administration. Iran. Biomed. J. 23, 287–296. Díaz-García, A., Ruiz-Fuentes, J.L., Fri� on-Herrera, Y., Yglesias-Rivera, A., Riquenez Garlobo, Y., Rodríguez S� anchez, H., Rodríguez Aurrecochea, J.C., L� opez Fuentes, L. X., 2019. Rhopalurus junceus scorpion venom induces antitumor effect in vitro and in vivo against a murine mammary adenocarcinoma model. Iran. J. Basic Med. Sci. 22, 759–765. https://doi.org/10.22038/ijbms.2019.33308.7956. Diaz-Garcia, A., Ruiz-Fuentes, J.L., Rodriguez-Sanchez, H., Fraga Castro, J.A., 2017. Rhopalurus junceus scorpion venom induces apoptosis in the triple negative human breast cancer cell line MDA-MB-231. J. Venom Res. 8, 9–13. Diaz-Garcia, A., Ruiz-Fuentes, J.L., Yglesias-Rivera, A., Rodriguez-Sanchez, H., Riquenes Garlobo, Y., Fleitas Martinez, O., Fraga Castro, J.A., 2015. Enzymatic analysis of venom from Cuban scorpion Rhopalurus junceus. J. Venom Res. 6, 11–18. Ding, J., Chua, P.J., Bay, B.H., Gopalakrishnakone, P., 2014. Scorpion venoms as a potential source of novel cancer therapeutic compounds. Exp. Biol. Med. 239, 387–393. https://doi.org/10.1177/1535370213513991. Dorce, A.L., Bellot, R.G., Dorce, V.A., Nencioni, A.L., 2009. Effects of prenatal exposure to Tityus bahiensis scorpion venom on rat offspring development. Reprod. Toxicol. 28, 365–370. https://doi.org/10.1016/j.reprotox.2009.04.008. EUROPEAN-PARLIAMENT, 2010. Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the Protection of Animals Used for Scientific Purposes Text with EEA Relevance. García-G� omez, B.I., Coronas, F.I.V., Restano-Cassulini, R., Rodríguez, R.R., Possani, L.D., 2011. Biochemical and molecular characterization of the venom from the Cuban scorpion Rhopalurus junceus. Toxicon 58, 18–27. https://doi.org/10.1016/J. TOXICON.2011.04.011. Grupo M�edico LifEscozul, 2018. Escozul conozca los resultados obtenidos por tipo de cancer. Escozul [WWW Document]. URL. https://escozul-cuba.com/resultados-con -escozul/. accessed 1.1.20. Gwee, M.C., Cheah, L.S., Gopalakrishnakone, P., 1995. Involvement of the L-argininenitric oxide synthase pathway in the relaxant responses of the rat isolated anococcygeus muscle to a scorpion (Leiurus quinquestriatus quinquestriatus) venom. Toxicon 33, 1141–1150. https://doi.org/10.1016/0041-0101(95)00064-s.
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Contents lists available at ScienceDirect
Toxicon journal homepage: http://www.elsevier.com/locate/toxicon
Safety evaluation of the venom from scorpion Rhopalurus junceus: Assessment of oral short term, subchronic toxicity and teratogenic effect Alicia Lagarto a, *, 1, Viviana Bueno a, 1, María R. P�erez b, 2, Caridad C. Rodríguez b, 2, Irania Guevara b, 2, Odalys Vald�es a, 1, Addis Bellma a, 1, Tatiana Gabilondo a, 1, � n a, 1 Alejandro S. Padro a b
Drug Research and Development Center, CIDEM, Ave 26 N 1605 entre Ave Boyeros y Calzada de Puentes Grandes, La Habana, Cuba Laboratories of Biopharmaceuticals and Chemistries Productions, LABIOFAM, Ave Independencia Km 16 1/2 Mulgoba, Boyeros, La Habana, Cuba
A R T I C L E I N F O
A B S T R A C T
Keywords: Rhopalurus junceus Scorpion venom Oral toxicity Teratogenic Toxicological profile
Rhopalurus junceus is the most common scorpion in Cuba and the venom is often used as a natural product for anti-cancer therapy. Despite this, no study has been published concerning its toxicological profile. The aim of the study was characterizing the short-term, subchronic toxicity and the teratogenic potential of Rhopalurus junceus scorpion venom by oral route in mice. Short-term oral toxicity was test in both sexes NMRI mice that received 100 mg/kg/day of scorpion venom extract for 28 days. For the subchronic study, mice were administered with three doses (0.1, 10, and 100 mg/kg) by oral route for 90 days. Teratogenic potential was tested in pregnant mice administered from day 6–15 post conception. Significant differences were observed in body weight and food intake of animal treated for short-term and subchronic assays. Variations in serum urea and cholesterol were observed after 90 days oral treatment. Spontaneous findings not related to the treatment were reveal in histology evaluation. Exposure in pregnant mice did not produce maternal toxicity. Signs of embryo-fetal toxicity were not observed. The current study provides evidence that exposure to low or moderate dose of Rhopalurus junceus scorpion venom by oral route did not affect health of animals and has low impact on reproductive physiology.
1. Introduction
It has previously been reported that soluble venom of this arachnid is not toxic to mice, injected intraperitoneally at doses up to 200 μg/20 g body weight, but it is deadly to insects at doses of 10 μg per animal �mez et al., 2011). The venom causes typical alpha and (García-Go beta-effects on Na þ channels, when assayed using patch-clamp tech niques in neuroblastoma cells in vitro. It also affects Kþ currents con ducted by ERG (ether-a-go-go related gene) channels. The soluble venom was shown to display phospholipase, hyaluronidase and �mez et al., 2011). Furthermore, the anti-microbial activities (García-Go venom from Rhopalurus junceus induces selective and differential anti cancer effect against epithelial cancer cells (Diaz-Garcia et al., 2013) and produced selective cytotoxic effect associated with its apoptosis-inducing effect through the mitochondrial pathway in human breast carcinoma cell line (Diaz-Garcia et al., 2017).
Scorpion venom contains various groups of compounds that exhibit a wide range of biological properties and actions in cells. The general composition and expression level of scorpion venom depends on genetic variation and geographical environment (Song et al., 2012). The scor pion Rhopalurus junceus is an endemic species from Cuba belonging to Buthidae family. This scorpion is widespread and there is no report of scorpionism from this or other species in the country. For this reason, they are not considered dangerous to humans. For a long time, a diluted preparation of venom from Rhopalurus junceus has been used in Cuban traditional medicine for treatment of some illnesses, including cancer, and has shown beneficial effects for some people (Diaz-Garcia et al., 2013; Hern� andez et al., 2009).
Abbreviations: LABIOFAM, Laboratories of Biopharmaceuticals and Chemistries Productions; CENPALAB, Laboratory Animal National Centre; SVE, scorpion venom extract. * Corresponding author. Ave 26 N1605 entre Ave Boyeros y Calzada de Puentes Grandes, Havana City, Cuba. E-mail addresses: [email protected] (A. Lagarto), [email protected] (V. Bueno). 1 [email protected] 2 [email protected] https://doi.org/10.1016/j.toxicon.2020.02.002 Received 21 October 2019; Received in revised form 28 January 2020; Accepted 3 February 2020 Available online 11 February 2020 0041-0101/© 2020 Elsevier Ltd. All rights reserved.
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The use of Rhopalurus junceus scorpion venom in humans needs a safety evaluation for oral route of administration. Available data are insufficient to support the safety of this preparation by oral route. The overall objective of this investigation was to characterize the short-term, subchronic toxicity and the teratogenic potential of Rhopalurus junceus scorpion venom by oral route in mice. An additional aim was to identify the no-observed-adverse-effect levels (NOAELs) for subchronic dosing and exposure during organogenesis period.
Spectronic Genesys 2. 2.4. Subchronic toxicity test Mice were randomized to four groups of each sex (4–5 weeks of age, 18–20 g of body weight, n ¼ 10 per group). Animals were exposed to SVE by gavage for 90 days as described above. The dosages delivered were 0.1, 10 and 100 mg/kg/day. Animals of control groups did not received treatment. Animals were monitored for body weight, food consumption, and behavioral and clinical signs as described in the shortterm toxicity study. At the completion of the subchronic study, blood samples were collected as described above and serum obtained for he matological and biochemical analyses (OECD, 1998). After collection of blood samples, mice were euthanized by exsan guination. Selected organs for weight were liver, kidneys, adrenals, spleen, thymus, brain, heart, ovaries, epididimos and testes. Selected organs (heart, kidneys, liver, spleen, brain, lungs, esophagus, stomach, intestines, thymus, adrenals, thyroid, parathyroid, trachea, pancreas, femur, bone marrow, salivary glands, cervical ganglion, epididimos, gonads, prostate, uteru, ovaries, seminal bladder and femoral muscle) were removed, fixed, sectioned, and stained for histopathological ex amination (OECD, 1998). A satellite group of 10 animals per sex (5 control and 5 treated) was used in the top dose group for observation, after the treatment period, for reversibility or persistence of any toxic effects. In these groups blood and organs were taken 28 days after the end of the 90 day treatment period. Additionally, hematological and clinical biochemistry analysis were made with animals before dosing to establish the range of control value (OECD, 1998).
2. Materials and methods 2.1. Test substances Adults Rhopalurus junceus scorpions were maintained in individual plastic cages in laboratories belonging to Laboratories of Bio pharmaceuticals and Chemistries Productions (LABIOFAM). Venom from scorpions kept alive in the laboratory was extracted by electrical stimulation. Venom was dissolved in distilled water and centrifuged at 15000�g for 15min. The supernatant was filtered by using a 0.2 μm syringe filter and stored at 20 � C until used. The protein concentration was calculated by the Lowry modified method. A solution with a protein concentration of 8–11 mg/ml was used in the studies. 2.2. Animals Animal care was performed in conformity with the Guide for the Care and Use of Laboratory Animals (National-Research-Council and Press, 2011). Healthy both sexes NMRI albino mice weighing 22 � 2 g were used in the studies. Animals were obtained from the Laboratory Animal National Centre (CENPALAB), Havana, Cuba and were randomly assigned to dosage groups. Animals was were housed together by sex in polycarbonate cages in a light- and humidity-controlled biohazard suite (24 � 2 � C; 55 � 5% relative humidity), with a 12-h light-dark cycle, and free access to drinking water and a standard laboratory diet CMO1000 (CENPALAB). Experiments were conducted in accordance with UE Directive 2010/63/EU for animal experiments (EUROPEAN-PARLIA MENT, 2010). The experimental protocols were approved by the Insti tutional Ethical Committee.
2.5. Evaluation of teratogenic effect Mature and healthy NMRI mice (female of 25 � 2 g and male of 30 � 2 g) were housed in plastic cages and quarantined for one week before mating. Four females were cohabited overnight with a male and examined for the presence of vaginal plug in their vagina. The day that vaginal plug was detected was defined as gestational day 0. Pregnant mice were randomized in control and treated groups (20 animals per group) and administered by intragastric intubation (gavage) at doses of 0.1, 10 and 100 mg/kg/day on gestational days 6–15 (ICH, 2005). Pregnant mice were observed daily for clinical signs and mortality. Body weight and food intake were monitored three times a week. Dams were killed by diethyl ether anesthesia and necropsy was performed on gestational day 18. Blood was taken from orbital sinus and examined for hematology (hemoglobin, hematocrit, erythrocyte and leukocyte count) and biochemical parameters (glucose, total cholesterol, aspartate aminotransferase (AST), and alkaline phosphatase). Macroscopic ex amination of organs was conducted for all dams, and organs with macroscopic damage were preserved for histological evaluation. Dams and fetus were examined as described previously (Burdan et al., 2005; Claudio et al., 1999; Lagarto et al., 2013).
2.3. Short-term toxicity test The study was designed to monitor the effect of a top dose on clinical signs, body weight, biochemical and hematological parameters previous to 90 days toxicity study. Mice were randomized to two groups of each sex (5–6 weeks of age, 24–27 g of body weight, n ¼ 5 per group). Ani mals were exposed to 100 mg/kg of scorpion venom extract (SVE) by gavage for a 28-day period. The dose selected was the top dose possible according to the nature of the substance and was ten times the thera peutic dose in preclinical studies. Administration volume was adjusted every 7 days according to body weight change. Another 5 mice of each sex were assessed for control without treatment. Animals were moni tored weekly for body weight and food consumption. Both behavior and clinical signs were monitored daily. Blood samples were collected in fasted overnight animals under anesthesia of Tiopental (40 mg/kg) on day 28 of dosing for hematology and clinical biochemistry. Gross pa thology was performed to all animal for organs and tissues (OECD, 2008). Hematology included determination of hematocrit by micro hematocrit capillaries, hemoglobin concentration by diagnostic kit produce by Biologic Products Inc. Havana, Cuba, erythrocyte and leukocyte count in Neubauer chamber, and differential leukocyte count by extension in microscopy slides. Clinical biochemistry included glucose, total cholesterol, creatinine, urea, albumin, alanine amino transferase (ALT) and alkaline phosphatase. The measures were made with diagnostic kit produced by Biologic Products Inc. Havana, Cuba. The absorbance values were determined in a spectrophotometer
2.6. Statistics Results were expressed as the mean � SEM. All statistical analysis was assessed using the GraphPad Prism Version 7 (GraphPad Software, San Diego, California, USA). Each test group was compared with control. Bartlett’s test was applied to determine whether the variance was ho mogeneous or not. When the variance was homogenous, one-way ANOVA was applied. When the variance was not homogenous, the Kruskal–Wallis test (nonparametric ANOVA) was performed. Statistical differences in the means among the groups were analyzed by means of Dunnett’s multiple comparison test. The parameters of developmental toxicity in fetuses were expressed with the litter as basic unit. Incidences of visceral and skeletal malformations/variations were analyzed by means of Fisher exact probability. A Two-sided analysis with a p-value of 60
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in urea and cholesterol levels in animals treated with SVE was reversible (Table 2). Similar histological findings were observed in treated and control animals.
0.05 or 0.01 was performed to determine statistical significance. 3. Results 3.1. Short-term toxicity test
3.3. Evaluation of teratogenic effect
Signs of toxicity were not observed during the experimental period in treated groups. Body weight trend were affected in treated groups. In male treated animals, body weight was significantly lower than control group throughout the study. In female, significant diference was observed until the second week of the experiment. Significantly lower body weight gain was observed in both sexes treated animal. In female treated animals, food consumption was significantly lower than control group (Fig. 1). No statistical differences between treated and control groups were observed in biochemical and hematological parameters.
Maternal toxicity: No evident clinical signs were observed in any dam during the study. There were no differences between the treated groups and controls for the maternal weight gain, maternal body weight in crease during treatment, gravid uterine weight, maternal net weight gain, food intake, or hematology and biochemical parameters (Table 3). Macroscopic finding of organs and tissues were not observed in dams. Embryo-fetal Toxicity: Statistical difference was not observed in number of implantation sites, corpora lutea, live and dead fetuses, as well as for resorptions, pre-implantation and post-implantation loss between SVE-exposed groups and the control (Table 4). Types and fre quencies of external abnormalities observed in fetuses are shown in Table 5. Changes affected paws and head. No statistical difference was observed in frequency of external variations and malformations compared to control group. Skeletal and visceral abnormalities were evaluated in high dose exposed fetuses (Table 6). The visceral malformations and variations observed in the present study were limited to the brain at low incidence. Three litters in control and one litter in high dose group miss for skeletal examination due to damage during the fixation process. All skeletal changes were limited to the axial skeleton and lumbar arch. These findings were spontaneous and unrelated to the drug. Oral treatment with SVE at 100 mg/kg on gestational day 6–15 neither induced skeletal abnormalities nor affected visceral morphology.
3.2. Subchronic toxicity test Signs of toxicity and mortality were not observed during the 90 day experimental period in treated groups. A significant variation in body weight trends was observed during the study period in both sexes (Fig. 2). In addition, food intake was significant affected in all doses in male. Low and high dose treated groups were the most affected. Food intake in medium dose group was significant decreased only at 6th and 7th weeks. In female, food intake was less affected than male. Significant differences were observed in low and medium dose groups only in a few weeks. Body weight gain (three doses) was significant affected in male. Dose effect relationship was assed for body weight gain by lineal regresion being significant for female (Fig. 3). No treatment-related changes were observed in hematological pa rameters tested during the study period. Clinical chemistry examination showed a decrease in urea level at 10 mg/kg/day (male) and 100 mg/ kg/day (both sexes). Additionally, cholesterol level was decrease after 90 days of oral treatment in male being significant at 100 mg/kg/day. The others biochemical parameters were not affected by dosing with SVE (Table 1). Any statistical variation was not observed in the relative organ weight (as % of total body weight) compared to control group. There was no treatment related histological changes in animals treated with high dose and control group in both sexes. Mandibular lymph node lymphoma, liver inflammation (hepatitis) and pneumonia were observed in one male of control group. In female control group, one animal with bronchitis and tracheitis was observed. In male treated group, pneumonia was observed in one animal. In female treated group, tracheitis, pneumonia, focal pancreatitis and liver microgranuloma were observed in one animal (Fig. 4). Satellite Group Results. Variations observed in body weight, weight gain and food intake in male treated animals remained significantly lower than controls 28-days after treatment (Fig. 5). Decrease observed
4. Discussion Scorpion and its products have been used as a traditional Chinese medicine for thousands of years. It has previously been reported that crude scorpion venom or isolated peptides from scorpion venom may inhibit the proliferation of cancer cells and induce cell apoptosis (Ding et al., 2014; Song et al., 2012). Scorpion venom contains peptides which exhibit anti-microbial properties. Kievit et al. reported that an ingre dient in the venom of the “death stalker” scorpion could help in gene therapy for effective treatment for brain cancer (Kievit et al., 2010). Rhopalurus junceus scorpion is the most common scorpion in Cuba. The venom showed cytotoxic effect in murine myeloma and rat prostate tumor cell lines (Hern� andez et al., 2009) and induces selective and differential anticancer effect against cancer cells (Diaz-Garcia et al., 2017, 2013). Enzymatic analysis of venom showed low enzymatic ac tivity, which could contribute to the low toxic potential of this scorpion venom (Diaz-Garcia et al., 2015). However, there was insufficient literature available of safety evaluation of products from scorpion venom and recent researches mainly focus on pharmacology evaluation and other relevant topics but none of them put emphasis on safety
Fig. 1. Body weight trends and gains and food intake during short-term toxicity test. The data are expressed as mean � SEM of 5 animals, *p < 0.05 **p < 0.01 ***p < 0.001 (significantly different from control). 61
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Fig. 2. Body weight trends and food intake during subchronic toxicity test in male (A–B) and female (C–D). The data are expressed as mean � SEM of 10 animals, *p < 0.05 **p < 0.01 ***p < 0.001 (significantly different from control).
Fig. 3. Body weight gain during subchronic toxicity test (A) and doses effect relationship (B). The data are expressed as mean � SEM of 10 animals, *p < 0.05 **p < 0.01 (significantly different from control).
evaluation. For safety evaluation, SVE was orally given at doses up to 100 mg/ kg/day to male and female mice. Our results indicate that oral exposure to SVE affect weight trends. In fact, a significant difference in weight gain was observed in male. Previous study indicate that the intravenous injection of toxin-γ from Tityus serrulatus scorpion venom induces a rapid, intense and sustained inhibition of gastric emptying (Bucaretchi et al., 1999). Sofer et al. reported that Leiurus quinquestriatus scorpion venom provoked gastrointestinal ischemia in pigs (Sofer et al., 1997) whereas Gwee et al. and Teixeira et al. respectively, demonstrated that L. quinquestriatus and T. serrulatus scorpion venoms were capable of relaxing rat isolated anococcygeus muscle and rabbit isolated corpus cavernosum via the local release of nitric oxide (Gwee et al., 1995; Teixeira et al., 1998). Pharmacokinetics and biodistribution of SVE in mice showed 30% of bioavailability by oral route and stomach, kidney, spleen and lung showed the major uptakes for 131I-labelel venom (Dia z-Garcia et al., 2019). These observations are important since a decrease
in gastrointestinal blood flow and the local release of nitric oxide may inhibit gastric smooth muscle cell activity (Chou, 1982; Shah et al., 2004). Thus, the effect on weight trends observed could be due to in hibition of gastric emptying by some components of SVE causing en hances satiety and decrease the food intake. Female mice were less affected than male. Female did not present significant alterations in weight gain and food intake was minimal affected by treatment. How ever, a dose effect ratio was observed in weight gain. Variations in body weight and food intake did not affect the integrity of organs and tissues and did not influence the health condition of the animals. Hematological parameters were not significant affected by sub chronic dosing with SVE. Significant decrease in serum urea was observed in both sexes treated animals. Common causes of decrease in urea concentration includes severe liver disease, low protein diet, severe polyuria/polydipsia and drugs. Absent of variations in liver enzymes and histopathological lesions excludes liver disease factor. On the other hand, variations in food intake could be associated with reduced urea 62
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production but the same dose groups for food intake and variations in urea concentration were not affected. In male animals, food consump tion was more affected in low and high dose groups than medium dose group. Serum urea variations were significant in medium and high dose groups. In female animals, were more evident that effects on food intake and urea are not related. However, variations in urea concentration could be related to the SVE oral exposure. High dose groups treated animals of both sexes and medium dose group in male animals were significant affected. SVE oral exposure up to 28 days did not affect urea concentrations at high dose and results of satellite groups in oral 90 days study showed that the effect on urea was reversible. The decrease in urea may be related to low protein absorption in the gastrointestinal tract or polyuria/polydipsia but further studies are required to clarify the causes. On the other hand, there is no evidence that the effect on urea concentration represent any functional impairment in treated animals and there is no related to a change in other parameters. The results are close to control range and the severity of the change is minimal. For that, we consider that the urea variation observed in treated animals does not constitute an adverse effect. Not significant decrease in creatinine concentration was observed in male treated animals. These results are within normal biological varia tion. Values are within control range used in the study and within reference values for this strain (Aleman et al., 2000). Variation in cholesterol observed was minimal and close to control range. This change did not affect health state of the animals and was reversible. For that, these variations are considered without pathological significance. Findings in histopathological exam were minimal and include very common lesions reported in mice. Tracheitis is a frequent finding of small foci of inflammatory cell infiltrates with low incidence, sporadic finding resulting from infections caused by gavage accidents. Bronchitis and pneumonia are especially common in females with small foci of inflammatory cells, predominantly lymphocytes. Pancreatitis as a small aggregates of lymphoid cells are common in mice. Liver inflammation (hepatitis) is a very common finding, up to 50% of CD-1 mice have small foci of both acute and chronic inflammatory cells scattered in the liver
Table 1 Results of biochemical parameters following 90-days exposure to SVE. Values represent the mean � SEM (n ¼ 10), **p < 0.01 significantly different from control. Treatment
Control
Scorpion venom extract
Doses (mg/kg/ day) Male Alkaline phosphatase (U/l) ALT (U/l)
0
0.1
10
100
146.1 � 15.0
125.7 � 11.1
137.1 � 12.7
118.6 � 8.9
109–183
Creatinine (μmol/l) Glucose (mmol/l)
63.9 � 6.4 4.4 � 0.5
Cholesterol (mmol/l) Albumin (g/l)
3.2 � 0.2 14.2 � 1.3
36.8 � 7.3 6.5 � 0.7 61.7 � 5.5 4.3 � 0.7 2.7 � 0.3 12.8 � 1.1
39.5 � 11.5 5.2 � 0.4** 46.0 � 3.4 4.9 � 0.2 2.5 � 0.2 13.8 � 0.7
23.5 � 6.1 5.4 � 0.7** 45.8 � 7.4 3.8 � 0.4 2.2 � 0.1** 14.5 � 0.8
13.6–42.4
Urea (mmol/l)
28.5 � 5.9 8.4 � 0.5
149.3 � 13.2
137.9 � 11.0
165.0 � 23.5
150.8 � 13.7
117–182
Creatinine (μmol/l) Glucose (mmol/l)
55.2 � 10.3 4.9 � 0.5
Cholesterol (mmol/l) Albumin (g/l)
1.9 � 0.4
24.5 � 5.5 5.1 � 0.5 44.2 � 5.9 4.4 � 0.9 1.2 � 0.1 12.6 � 0.6
26.0 � 6.0 6.2 � 0.7 47.4 � 5.2 4.6 � 0.3 1.9 � 0.1 14.8 � 0.6
18.2 � 3.8 3.3 � 0.2** 60.0 � 11.5 4.8 � 0.5 1.9 � 0.1 14.7 � 0.3
15.2–35.8
Urea (mmol/l)
25.5 � 4.2 6.2 � 1.0
Female Alkaline phosphatase (U/l) ALT (U/l)
13.0 � 0.8
Control Range
6.9–9.9 38.7–69.0 3.1–5.7 2.4–3.9 10.9–17.4
4.3–9.4 28.8–81.7 3.8–6.0 1.0–2.8 10.6–15.3
Fig. 4. Photomicrograph showing histological findings in representative animal. (A) Control lung with normal histology. (B) Pneumonia observed in female treated animals. (C) Control liver with normal histology. (D) Inflammation (hepatitis) with necrosis observed in one male control animal (hematoxylin and eosin stain). 63
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Fig. 5. Body weight trends and food intake during subchronic toxicity test in satellite group. The data are expressed as mean � SEM of 5 animals, *p < 0.05 **p < 0.01 ***p < 0.001 (significantly different from control). Table 2 Biochemical results of animals 28 days after 90-days exposure to SVE. Values represent the mean � SEM (n ¼ 5). Parameter
Urea (mmol/l) Cholesterol (mmol/ l)
Male
Table 3 Effect of SVE on maternal toxicity end points in mice. Values represent the mean � SEM (n ¼ 20).
Female
Control
100 mg/kg/ day
Control
100 mg/kg/ day
9.8 � 1.8 3.3 � 0.8
8.7 � 0.9 2.8 � 0.7
10.4 � 1.7 2.6 � 0.4
9.7 � 0.5 3.1 � 0.6
Treatment
Control
Scorpion venom extract
Dosage (mg/kg) No. of pregnant animals treated. No. of animals at term gestation with live fetuses. Initial body weight (GD 0/g�SEM).
0 20 20
0.1 21 20
10 20 20
100 20 20
26.2 � 0.5 2.3 � 0.4
27.0 � 0.5 2.1 � 0.3 12.5 � 0.4 9.2 � 0.6 23.9 � 0.8 16.9 � 0.5 7.0 � 0.6 5.4 � 0.2
26.1 � 0.4 2.4 � 0.4 12.9 � 0.5 9.0 � 0.6 24.4 � 0.8 16.5 � 0.8 7.5 � 0.5 5.6 � 0.1
25.6 � 0.4 1.8 � 0.3 12.5 � 0.4 8.4 � 0.6 22.7 � 0.8 16.1 � 0.6 6.6 � 0.5 5.4 � 0.2
11.1 � 0.8 39.1 � 1.5 5.57 � 0.6 4.52 � 0.6
10.7 � 0.6 40.0 � 1.1 5.70 � 0.5 5.75 � 0.7
9.5 � 0.8 39.8 � 1.3 5.59 � 0.4 5.04 � 0.7
9.4 � 0.9 39.7 � 1.6 5.76 � 0.4 3.76 � 0.3
4.2 � 0.4
3.9 � 0.2 3.6 � 0.3 73.0 � 3.6 85.9 � 4.2
4.1 � 0.3 3.4 � 0.4 77.1 � 4.3 84.8 � 4.5
3.9 � 0.3 2.7 � 0.3 76.9 � 4.7 91.9 � 5.4
Maternal body weight gain (GD 0–6/g�SEM). Maternal body weight gain (GD 6–15/g�SEM). Maternal body weight gain (GD 15–18/g� SEM). Maternal body weight gain (GD 0–18/g� SEM). Gravid uterine weight at term (g�SEM). Maternal net weight gain (g� SEM). Food intake (g/animal/day� SEM)
(Peckham, 2002). Considering the incidence and nature of the histo logical changes they are spontaneous and unrelated to the treatment. In the teratological study conducted in a single rodent species, adverse reproductive effects were not observed after the administration of three doses of SVE by oral gavage. Maternal toxicity was not observed because of exposure to the test substance by clinical observations, body weight gain, food intake, hematology and biochemical parameters and necropsy finding. Slight increase of pre-implantation loss was observed with 10 mg/kg treated animals. This change was without statistically significant and not related to the treatment because the SVE exposure began on day 6 post conception and the variations occurred before im plantation. External anomalies were similar for all experimental groups. Incidence of hemorrhage does not represent an anomaly and it is considered because of a functional disorder and thus not strictly devel opmental anomaly. For that reason, hemorrhage was classified as “Not Malformation” (Solecki et al., 2003). The total number of litters and fetuses with visceral and skeletal malformations and variations were similar in all experimental groups. We considered that these finding was spontaneous and unrelated to the drug. There are not studies in literature about the toxic effect of the venom from the scorpion Rhopalurus junceus administered by oral route in ro dents. It is known that the scorpion venom can cause malformations or behavioral defects to the offspring of mothers exposed to the venom during pregnancy. Previous reports reflected the effect of prenatal exposure to Tytus bahiensis scorpion venom. A moderate dose of this venom injected to pregnant rats was able to elicit alterations in physical and behavioral development in the offspring during the postnatal period
Hematology Hemoglobin (mmol/L� SEM) Hematocrit (%�SEM) Total erythrocyte count (cell/ mm3x106�SEM) Total leukocyte count (cell/mm3 x103�SEM) Biochemistry Glucose (mmol/L�SEM)
24.9 � 0.8 17.4 � 0.6 7.8 � 0.5 5.5 � 0.2
Total cholesterol (mmol/L�SEM)
3.5 � 0.3
AST (U/L�SEM)
78.5 � 6.2 93.4 � 4.1
Alkaline phosphatase (U/L�SEM)
64
13.7 � 0.6 9.0 � 0.5
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Table 4 Effect of SVE on embryo-fetal parameters in mice. Values represent the mean � SEM (n ¼ 20). Treatment
Control
Scorpion venom extract
Dosage (mg/kg) Litters with live fetuses. No. of corpora lutea (Mean � SEM)/litter. Mean � SEM of implantations/litter No. of implantations. No. of live fetuses (Mean � SEM)/litter. No. of dead fetuses (litters affect) No. of early resorptions (litters affect) No. of late resorptions (litters affect) Pre-implantations loss (%) Post-implantation loss (%) Mean placenta weight (g � SEM)/litter. Mean fetal body weight (g � SEM)/litter. 1. Male
0 20 12.8 � 0.4 12.6 � 0.4 251 11.6 � 0.5 1(1)
0.1 20 12.6 � 0.4 12.3 � 0.4 246 11.2 � 0.4 1(1)
10 20 12.6 � 0.4 11.5 � 0.6 230 10.8 � 0.5 1(1)
100 20 11.6 � 0.5 11.4 � 0.4 227 10.6 � 0.5 1(1)
11(10)
13(9)
7(6)
10(7)
6(3)
9(8)
5(4)
4(3)
1.95 7.17 0.14 � 0.01 1.29 � 0.03 1.31 � 0.03 1.24 � 0.05 3.0 � 0.5
2.77 9.35 0.13 � 0.006 1.30 � 0.02 1.30 � 0.02 1.28 � 0.02 3.0 � 0.5
8.73 6.09 0.13 � 0.006 1.33 � 0.02 1.32 � 0.02 1.34 � 0.03 3.5 � 0.5
1.73 7.05 0.13 � 0.008 1.31 � 0.03 1.32 � 0.03 1.29 � 0.03 2.9 � 0.4
2. Female Sex ratio (M/F).
Table 6 Visceral and skeletal defects following oral maternal exposure to SVE. Values represent the mean � SEM (n ¼ 20).
Treatment
Control
Scorpion venom extract 0.1 223 (20)
10 216 (20)
100 211 (20)
3(3) 1(1) 1(1) 0 1(1) 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0
0 0
0 0
2(2)
4(4)
3(3)
Control
Scorpion venom extract
Dosage (mg/kg) Number fetuses (litters) examined – Visceral Exam Visceral malformations No. of fetuses with malformations Cerebrum – Misshapen Visceral variations No. of fetuses with variations Brain - Hemorrhage Lateral cerebral ventricle – Dilated Number fetuses (litters) examined – Skeletal Exam Skeletal malformations No. of fetuses with malformations Lumbar arch - Misshapen Sternebra - Absent Xiphisternum - Absent Skeletal variations No. of fetuses with variations Rib - Misaligned Xiphisternum – Bipartite ossification Xiphisternum- Incomplete ossification Sternebra- Incomplete ossification Sternebra - Asymmetric
0 100 118 104(20) (20) Fetuses affected (litters affected) 0 1(1) 0 1(1) 3(3) 6(2) 3(3) 1(1) 0 5(1) 96(17) 101(19) 1(1)
4(3)
1(1) 0 0 2(2) 2(2) 0 0 0 0
1(1) 2(2) 1(1) 9(5) 2(2) 2(1) 2(2) 4(2) 1(1)
Published preliminary online data refer antitumoral activity in human (Grupo M� edico LifEscozul, 2018). Oral doses referred in traditional medicine are usually low, the use of the diluted preparation (6 ml four times a day) in patients with anal and rectal cancer after surgery has been reported (Poch Mulgado et al., 1999). Despite the traditional use, Rhopalurus junceus scorpion venom should be used with caution for the lack of safety and effective evaluation by oral route. In summary, 100 mg/kg/day orally was the subchronic NOAEL for Rhopalurus junceus scorpion venom, for effects other than variations in weight gain that did not affect the health state of the animals. The oral exposure in mice up to 100 mg/kg/day during organogenesis period did not induce significant maternal and embryo-fetal toxicity. The current study provides evidence that exposure to low or moderate dose of SVE by oral route did not affect health of animals and has low impact on reproductive physiology. Nevertheless, additional studies are necessary for a complete risk assessment and traditional use should be done with caution.
Table 5 External malformations and variations following oral maternal exposure to SVE. Values represent the mean � SEM (n ¼ 20). Dosage (mg/kg) 0 Number fetuses (number litters) 233 examined (20) External malformations No. of fetuses with malformations 8(2) Meningo-encephalocele 0 Exencephaly 1(1) Tongue - Absent 2(1) Hindlimb – Malrotated 0 Palate - Cleft 5(1) External variations No. of fetuses with variations 1(1) Palatal rugae - Absent 1(1) External not malformation (unclassified) Hemorrhage 5(4)
Treatment
CRediT authorship contribution statement Alicia Lagarto: Conceptualization, Methodology, Validation, Formal analysis, Writing - original draft, Writing - review & editing, Project administration. Viviana Bueno: Investigation, Data curation. �rez: Conceptualization, Methodology, Investigation, María R. Pe Writing - review & editing. Caridad C. Rodríguez: Conceptualization, Methodology, Investigation, Writing - review & editing. Irania Gue vara: Conceptualization, Methodology, Investigation, Writing - review �s: Investigation, Data curation. Addis Bellma: & editing. Odalys Valde Investigation, Data curation. Tatiana Gabilondo: Investigation, Data � n: Investigation, Supervision. curation. Alejandro S. Padro
(Barao et al., 2008; Dorce et al., 2009), while low subcutaneous doses of Tytus bahiensis venom did not cause maternal or clear fetal toxicity (Cruttenden et al., 2008). Rhopalurus junceus is known by the vulgar name of “Blue Scorpion” and a diluted solution of its venom known as “Escozul” (a trade mark) is an alternative drug used for pain relief, anti-inflammatory and cancer �mez et al., 2011; Yglesias-Rivera treatment, mainly in Cuba (García-Go and Díaz-García, 2018). There are several communications dealing with anticancer in vitro effect of the venom against cancer cell lines from epithelial origin without affecting normal cells, in the cervical cancer cell line HeLa (Diaz-Garcia et al., 2013; Yglesias-Rivera et al., 2019) and the metastatic breast cancer cell line, MDA- MB-231 (Diaz-Garcia et al., 2017). There are few effective assays performed in vivo, none orally. Significant tumor growth delay in the murine mammary adenocarci noma F3II and dose-dependent decrease of Ki-67 in tumor-bearing ani mals treated intraperitoneally at 0.2; 0.8; 3.2 mg/kg doses were reported recently (Díaz-García et al., 2019). However, oral treatment with Escozul in Cuban traditional medicine for cancer treatment has shown beneficial effects for some people.
References Aleman, C.L., Noa, M., Mas, R., Rodeiro, I., Mesa, R., Menendez, R., Gamez, R., Hernandez, C., 2000. Reference data for the principal physiological indicators in three species of laboratory animals. Lab. Anim. 34, 379–385. https://doi.org/ 10.1258/002367700780387741. Barao, A.A., Bellot, R.G., Dorce, V.A., 2008. Developmental effects of Tityus serrulatus scorpion venom on the rat offspring. Brain Res. Bull. 76, 499–504. https://doi.org/ 10.1016/j.brainresbull.2008.02.033. Bucaretchi, F., Vinagre, A.M., Chavez-Olortegui, C., Collares, E.F., 1999. Effect of toxin-g from Tityus serrulatus scorpion venom on gastric emptying in rats. Braz. J. Med. Biol. Res. 32, 431–434. https://doi.org/10.1590/s0100-879x1999000400009.
65
A. Lagarto et al.
Toxicon 176 (2020) 59–66 Hern� andez, O., Casado, I., et al., 2009. Evaluaci� on de la toxicidad in vitro del veneno del alacr� an Rophalurus junceus a trav�es de un ensayo celular. Rev. Cubana Invest. Biom�ed. 28. ICH, 2005. Detection of Toxicity to Reproduction for Medicinal Products & Toxicity to Male Fertility. S5(R2). Kievit, F.M., Veiseh, O., Fang, C., Bhattarai, N., Lee, D., Ellenbogen, R.G., Zhang, M., 2010. Chlorotoxin labeled magnetic nanovectors for targeted gene delivery to glioma. ACS Nano 4, 4587–4594. https://doi.org/10.1021/nn1008512. Lagarto, A., Bellma, A., Couret, M., Gabilondo, T., L� opez, R., Bueno, V., Guerra, I., Rodríguez, J., 2013. Prenatal effects of natural calcium supplement on Wistar rats during organogenesis period of pregnancy. Exp. Toxicol. Pathol. 65, 49–53. https:// doi.org/10.1016/j.etp.2011.05.009. National-Research-Council, Press, N.A., 2011. Guide for the Care and Use of Laboratory Animals, eighth ed. (Washington DC). OECD, 2008. Test No. 407: Repeated Dose 28-day Oral Toxicity Study in Rodents, OECD Guidelines for the Testing of Chemicals. OECD Publishing, Paris. https://doi.org/ 10.1787/9789264070684-en. OECD, 1998. Test No. 408: Repeated Dose 90-Day Oral Toxicity Study in Rodents, OECD TG 408. Guideline for the Testing of Chemicals. Peckham, J.C., 2002. Animal histopathology. In: Derelanko, M.J., Hollinger, M.A. (Eds.), Handbook of Toxicology. CRC Press, pp. 668–692. Poch Mulgado, E., Formental Planas, J.J., Alvarez Lambert, K.R., Bordier Chibas, M., Frometa Gomez, A.A., Andalia Ricardo, E., 1999. Uso Escozul ( Toxina De Alacran ) En Pacientes Operados Cancer. Anal. Guantanamo. Shah, V., Lyford, G., Gores, G., Farrugia, G., 2004. Nitric oxide in gastrointestinal health and disease. Gastroenterology 126, 903–913. https://doi.org/10.1053/j. gastro.2003.11.046. Sofer, S., Cohen, R., Shapir, Y., Chen, L., Colon, A., Scharf, S.M., 1997. Scorpion venom leads to gastrointestinal ischemia despite increased oxygen delivery in pigs. Crit. Care Med. 25, 834–840. https://doi.org/10.1097/00003246-199705000-00020. Solecki, R., Bergmann, B., Burgin, H., Buschmann, J., Clark, R., Druga, A., Van Duijnhoven, E.A., Duverger, M., Edwards, J., Freudenberger, H., Guittin, P., Hakaite, P., Heinrich-Hirsch, B., Hellwig, J., Hofmann, T., Hubel, U., Khalil, S., Klaus, A., Kudicke, S., Lingk, W., Meredith, T., Moxon, M., Muller, S., Paul, M., Paumgartten, F., Rohrdanz, E., Pfeil, R., Rauch-Ernst, M., Seed, J., Spezia, F., Vickers, C., Woelffel, B., Chahoud, I., 2003. Harmonization of rat fetal external and visceral terminology and classification. Report of the fourth workshop on the terminology in developmental toxicology, berlin, 18-20 April 2002 Reprod. Toxicol. 17, 625–637. Song, X., Zhang, G., Sun, A., Guo, J., Tian, Z., Wang, H., Liu, Y., 2012. Scorpion venom component III inhibits cell proliferation by modulating NF-kappaB activation in human leukemia cells. Exp. Ther. Med. 4, 146–150. https://doi.org/10.3892/ etm.2012.548. Teixeira, C.E., Bento, A.C., Lopes-Martins, R.A., Teixeira, S.A., von Eickestedt, V., Muscara, M.N., Arantes, E.C., Giglio, J.R., Antunes, E., de Nucci, G., 1998. Effect of Tityus serrulatus scorpion venom on the rabbit isolated corpus cavernosum and the involvement of NANC nitrergic nerve fibres. Br. J. Pharmacol. 123, 435–442. https://doi.org/10.1038/sj.bjp.0701623. Yglesias-Rivera, A., Díaz-García, A., 2018. Effect of Rhopalurus junceus scorpion venom on inflammation-related cytokines in healthy BALB/C mice. Ann. Microbiol. Immunol. 1, 1007. Yglesias-Rivera, A., Rodríguez-S� anchez, H., Díaz-García, A., 2019. Synergistic effect of Rhopalurus junceus scorpion venom combined with conventional cytostatics in cervical cancer cell line HeLa. J. Pharm. Pharmacogn. Res. 7, 67–76.
Burdan, F., Szumilo, J., Dudka, J., Klepacz, R., Blaszczak, M., Solecki, M., Korobowicz, A., Chalas, A., Klepacki, J., Palczak, M., Zuchnik-Wrona, A., HadalaKis, A., Urbanowicz, Z., Wojtowicz, Z., 2005. Morphological studies in modern teratological investigations. Folia Morphol. (Warsz) 64, 1–8. Chou, C.C., 1982. Relationship between intestinal blood flow and motility. Annu. Rev. Physiol. 44, 29–42. https://doi.org/10.1146/annurev.ph.44.030182.000333. Claudio, L., Bearer, C.F., Wallinga, D., 1999. Assessment of the U.S. Environmental Protection Agency methods for identification of hazards to developing organisms, Part II: the developmental toxicity testing guideline. Am. J. Ind. Med. 35, 554–563. https://doi.org/10.1002/(sici)1097-0274(199906)35:6<554::aid-ajim2>3.0.co;2-x. Cruttenden, K., Nencioni, A.L., Bernardi, M.M., Dorce, V.A., 2008. Reproductive toxic effects of Tityus serrulatus scorpion venom in rats. Reprod. Toxicol. 25, 497–503. https://doi.org/10.1016/j.reprotox.2008.04.011. Diaz-Garcia, A., Morier-Diaz, L., Frion-Herrera, Y., Rodriguez-Sanchez, H., CaballeroLorenzo, Y., Mendoza-Llanes, D., Riquenes-Garlobo, Y., Fraga-Castro, J.A., 2013. In vitro anticancer effect of venom from Cuban scorpion Rhopalurus junceus against a panel of human cancer cell lines. J. Venom Res. 4, 5–12. Diaz-Garcia, A., Pimentel Gonzalez, G., Basaco Bernabeu, T., Rodriguez Aurrecochea, J. C., Rodriguez Sanchez, H., Sanchez Monzon, I., Hernandez Gomez, M., Rodriguez Torres, C., Regla Rodriguez Capote, M., Guevara Orellanes, I., 2019. Pharmacokinetics and biodistribution of Rhopalurus junceus scorpion venom in tumor-bearing mice after intravenous and oral administration. Iran. Biomed. J. 23, 287–296. Díaz-García, A., Ruiz-Fuentes, J.L., Fri� on-Herrera, Y., Yglesias-Rivera, A., Riquenez Garlobo, Y., Rodríguez S� anchez, H., Rodríguez Aurrecochea, J.C., L� opez Fuentes, L. X., 2019. Rhopalurus junceus scorpion venom induces antitumor effect in vitro and in vivo against a murine mammary adenocarcinoma model. Iran. J. Basic Med. Sci. 22, 759–765. https://doi.org/10.22038/ijbms.2019.33308.7956. Diaz-Garcia, A., Ruiz-Fuentes, J.L., Rodriguez-Sanchez, H., Fraga Castro, J.A., 2017. Rhopalurus junceus scorpion venom induces apoptosis in the triple negative human breast cancer cell line MDA-MB-231. J. Venom Res. 8, 9–13. Diaz-Garcia, A., Ruiz-Fuentes, J.L., Yglesias-Rivera, A., Rodriguez-Sanchez, H., Riquenes Garlobo, Y., Fleitas Martinez, O., Fraga Castro, J.A., 2015. Enzymatic analysis of venom from Cuban scorpion Rhopalurus junceus. J. Venom Res. 6, 11–18. Ding, J., Chua, P.J., Bay, B.H., Gopalakrishnakone, P., 2014. Scorpion venoms as a potential source of novel cancer therapeutic compounds. Exp. Biol. Med. 239, 387–393. https://doi.org/10.1177/1535370213513991. Dorce, A.L., Bellot, R.G., Dorce, V.A., Nencioni, A.L., 2009. Effects of prenatal exposure to Tityus bahiensis scorpion venom on rat offspring development. Reprod. Toxicol. 28, 365–370. https://doi.org/10.1016/j.reprotox.2009.04.008. EUROPEAN-PARLIAMENT, 2010. Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the Protection of Animals Used for Scientific Purposes Text with EEA Relevance. García-G� omez, B.I., Coronas, F.I.V., Restano-Cassulini, R., Rodríguez, R.R., Possani, L.D., 2011. Biochemical and molecular characterization of the venom from the Cuban scorpion Rhopalurus junceus. Toxicon 58, 18–27. https://doi.org/10.1016/J. TOXICON.2011.04.011. Grupo M�edico LifEscozul, 2018. Escozul conozca los resultados obtenidos por tipo de cancer. Escozul [WWW Document]. URL. https://escozul-cuba.com/resultados-con -escozul/. accessed 1.1.20. Gwee, M.C., Cheah, L.S., Gopalakrishnakone, P., 1995. Involvement of the L-argininenitric oxide synthase pathway in the relaxant responses of the rat isolated anococcygeus muscle to a scorpion (Leiurus quinquestriatus quinquestriatus) venom. Toxicon 33, 1141–1150. https://doi.org/10.1016/0041-0101(95)00064-s.
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