Evaluation of the cytotoxicity, oral toxicity, genotoxicity, and mutagenicity of the latex extracted from Himatanthus drasticus (Mart.) Plumel (Apocynaceae)

Journal Pre-proof Evaluation of the cytotoxicity, oral toxicity, genotoxicity, and mutagenicity of the latex extracted from Himatanthus drasticus (Mart.) Plumel (Apocynaceae) Danielle Feijó de Moura, Tamiris Alves Rocha, Dayane de Melo Barros, Marllyn Marques da Silva, Maria Aparecida da Conceição de Lira, Talita Giselly dos Santos Souza, Camila Joyce Alves da Silva, Francisco Carlos Amanajás de Aguiar, Júnior, Cristiano Aparecido Chagas, Noemia Pereira da Silva Santos, Ivone Antônia de Souza, Renata Mendonça Araújo, Rafael Matos Ximenes, René Duarte Martins, Márcia Vanusa da Silva PII:

S0378-8741(19)34076-0

DOI:

https://doi.org/10.1016/j.jep.2020.112567

Reference:

JEP 112567

To appear in:

Journal of Ethnopharmacology

Received Date: 11 October 2019 Revised Date:

8 January 2020

Accepted Date: 8 January 2020

Please cite this article as: de Moura, Danielle.Feijó., Rocha, T.A., Barros, D.d.M., da Silva, M.M., de Lira, Maria.Aparecida.da.Conceiçã., dos Santos Souza, T.G., da Silva, C.J.A., de Aguiar Júnior., , Francisco.Carlos.Amanajá., Chagas, C.A., da Silva Santos, N.P., de Souza, Ivone.Antô., Araújo, Renata.Mendonç., Ximenes, R.M., Martins, René.Duarte., da Silva, Má.Vanusa., Evaluation of the cytotoxicity, oral toxicity, genotoxicity, and mutagenicity of the latex extracted from Himatanthus drasticus (Mart.) Plumel (Apocynaceae), Journal of Ethnopharmacology (2020), doi: https:// doi.org/10.1016/j.jep.2020.112567. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2020 Published by Elsevier B.V.

Evaluation of the cytotoxicity, oral toxicity, genotoxicity, and mutagenicity of the latex extracted from Himatanthus drasticus (Mart.) Plumel (Apocynaceae)

Danielle Feijó de Mouraa*, Tamiris Alves Rochaa, Dayane de Melo Barrosb, Marllyn Marques da Silvab, Maria Aparecida da Conceição de Lirab, Talita Giselly dos Santos Souzab, Camila Joyce Alves da Silvac, Francisco Carlos Amanajás de Aguiar Júniorb, Cristiano Aparecido Chagasb, Noemia Pereira da Silva Santosb, Ivone Antônia de Souzac, Renata Mendonça Araújod, Rafael Matos Ximenesc*, René Duarte Martinsb, Márcia Vanusa da Silvaa,e

a

Departamento de Bioquímica, Universidade Federal de Pernambuco, Recife, Brazil.

b

Centro Acadêmico de Vitória, Universidade Federal de Pernambuco, Brazil.

c

Departamento de Antibióticos, Universidade Federal de Pernambuco, Recife, Brazil.

d

Instituto de Química, Universidade Federal do Rio Grande do Norte, Natal, Brazil.

e

Núcleo de Bioprospecção da Caatinga, Instituto Nacional do Semiárido, Paraíba, Brazil.

*Corresponding authors: DFM ([email protected]) and RMX ([email protected]).

Abstract Ethnopharmacological relevance: Himatanthus drasticus is a tree popularly known as janaguba. Endemic to Brazil, it is found in the Cerrado and Caatinga biomes, rock fields, and rainforests. Janaguba latex has been used in folk medicine for its antineoplastic, anti-inflammatory, analgesic, and antiallergic activities. However, studies investigating the safety of its use for medicinal purposes are limited. Aim of the study: This study aimed to evaluate the toxicity of the latex extracted from H. drasticus.

Materials and methods: The latex was extracted from H. drasticus specimens by removing a small area of bark (5 x 30 cm) and then dissolving the exudate in water and lyophilizing it. Phytochemical screening was performed by TLC and GC-MS, protein, and carbohydrate levels. Cell viability was performed by the MTT method. Acute oral toxicity, genotoxicity, and mutagenicity assays were performed in mice. Results: TLC showed the presence of saponins and reducing sugars, as well as steroids and terpenes. The GC-MS analysis of the nonpolar fraction identified lupeol acetate, botulin, and α/β-amyrin derivatives as the major compounds. The latex was toxic to S180 cells at 50 and 100 µg/mL. No signals of toxicity or mutagenicity was found in mice treated with 2,000 mg/kg of the latex, but genotoxicity was observed in the Comet assay. Conclusions: H. drasticus latex showed toxicity signals at high doses (2,000 mg/kg). Although the latex was not mutagenic to mice, it was genotoxic in the Comet assay in our experimental conditions. Even testing a limit dose of 2,000 mg/kg, which is between 10 to 35-fold the amount used in folk medicine, caution must be taken since there is no safe level for genotoxic compounds exposure. Further studies on the toxicological aspects of H. drasticus latex are necessary to elucidate its possible mechanisms of genotoxicity.

Keywords: traditional medicine, Brazilian plants, phytochemistry, caatinga, janaguba

1. Introduction The use of medicinal plants in the Brazilian territory plays an important role in primary health care, especially in communities without access to modern medicine. Plants can be utilized both in natura, as teas, and in the manufacture of herbal medicines (Horn and Vargas, 2008). However, the use of plants in most cases is performed empirically, i.e. without scientific proof of the therapeutic efficacy and knowledge of possible adverse effects of plant consumption (Silveira and Sá et al., 2003). Among the plant species, Himatanthus drasticus (Mart.) Plumel (Apocynaceae), popularly known as janaguba, is a medium-sized tree that has been widely used by the Brazilian population due to its potential advantageous properties, a phenomenon that has piqued the interest of the scientific community. The latex of H. drasticus (known as janaguba “milk”) has been recommended for its anti-inflammatory and immunestimulating properties and is mostly used to treat cancer (Colares et al., 2008; Mousinho et al., 2011). An ethnobotanical survey about janaguba milk uses among practitioners in Ceará, Brazil, showed the crude latex is diluted to 20% in water to give the janaguba milk, which is consumed (15-150 mL/day) up to 3 months (Soares et al., 2015). Although there are few scientific reports regarding the chemical composition of H. drasticus latex, several terpenes have been identified and shown to have biological properties such as gastroprotective (Colares et al., 2008), anti-inflammatory (Lucetti et al., 2010), bactericidal, fungicidal, antiviral, analgesic and antiallergic properties (Patocka, 2003). Further, a report has shown that they also have antitumor activity (Mousinho et al., 2011). In view of the widespread empirical use of medicinal plants and a relatively small amount of scientific discoveries demonstrating the toxicity profile of plant species, the aim of this study was to evaluate the phytochemical profile and toxicological safety of H. drasticus.

2. Materials and methods 2.1. Botanical material The latex was collected from H. drasticus specimens in the Araripe Environmental Protection Area (Araripe APA, Crato-Ceará; 7°23’8424’’S and 39°46’8226’’O) through the removal of a small area of the stem bark (5 x 30 cm). The exudate was removed with distillated water and lyophilized. For the biological assays, the latex was dissolved in DMSO and then reconstituted in PBS (maximum 10% DMSO). A voucher specimen was deposited in the Herbarium of the Agronomic Institute of Pernambuco (IPA nº 92,408).

2.2. Phytochemical analysis The qualitative phytochemical analysis of the latex was performed by thin-layer chromatography (TLC) using silica gel chromatography plates 60 F254 (Merck, Germany) to verify the presence of flavonoids, phenylpropanoids, triterpenes, phytosterols, saponins, monoterpenes, sesquiterpenes, alkaloids, coumarins, quinones, proanthocyanidins, hydrolysable tannins, and reducing sugars. The standards, solvent systems, and chemical developers are described by Santos et al. (2018). Carbohydrate levels were measured using the phenol-acid method using the principle of total sugar according to Dubois (1956) and reducing sugars were measured using the 3,5dinitrosalicylic acid (DNS) method according to Miller (1959). Finally, the amount of protein in the latex was evaluated according to Lowry et al. (1951).

2.3 GC-MS analysis The chemical composition of the nonpolar fraction from the latex was analyzed by gas chromatography coupled to an ISQ Series Mass Spectrometer (Thermo Scientific™, MA, USA), using NST-5MS column (30 m x 25 mm x 10 µm). The

analyses were run at constant column flow of 1.0 mL/min and split injection mode. The temperature of the injector and detector were both 220 °C, and the transfer line was 300 °C. The heating ramp was 80 to 170 °C at 5 °C/min for 18 min and finally 170 to 300°C at 1°C/min for 130 min. Aliquots of 1 µL of a solution prepared by dissolving 1 mg of the nonpolar fraction in 1.0 mL of dichloromethane were injected. The scan range of the mass spectrometer was 40 to 600 m/z. The identification of the compounds was performed by comparison of the mass spectra obtained with the equipment database using the NIST library.

2.4 Animals Eight-week-old adult male Swiss mice from the Keizo Asami Immunopathology Laboratory (LIKA) of the Federal University of Pernambuco (UFPE) were used. The animals were kept in an environmentally controlled room at 22 ± 2 ºC, with relative air humidity of 50 ± 5% and 12h light-dark cycle, and free access to food and water. All animal procedures were approved by the Ethics Committee on Animal Use of the Federal University of Pernambuco (process nº 23076.008387/2015-59).

2.5 Cell lines and culture conditions Human cervical epithelial carcinoma (HeLa), Ehrlich ascites carcinoma (EAC) and Sarcoma-180 cells were used for experiments. HeLa cells were cultured in Dulbecco's modified eagle medium (DMEM), supplemented with fetal bovine serum (10%) and penicillin-streptomycin (1%), at 37 °C and 5% CO2. EAC is a derivative of murine breast adenocarcinoma and S-180 is a malignant, heterogeneous strain of mouse tumor cells of mesodermal origin (Debnath et al, 2017), both were in the intraperitoneal cavity of Swiss mice (Mus musculus) in their ascitic form (Mallick et al., 2015). After puncture, cells were quantified (106 cells/mL) for the cell viability tests using the MTT

method [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] as described by Mosmann (1983).

2.6 Isolation of murine peritoneal macrophages To isolate the murine peritoneal macrophages, 1 mL of 3.8% sterile thioglycolate medium was injected into the peritoneal cavity of 3 mice. After 72 h, the cells were collected using cold PBS and then centrifuged and resuspended in DMEM supplemented with antibiotics (Montoya et al, 2019).

2.7 Cytotoxicity Assay The MTT assay was used to determine the effects of H. drasticus latex on cell viability (Mosmann, 1983). Cells (Sarcoma-180, EAC, HeLa, and MPM) were seeded into 96-well plates at a density of 1 x 105– 1 x 106 cells/mL and after a 24 h incubation period, they were exposed to latex for 48 h at final concentrations of 12.5, 25, 50 and 100 µg/mL (Aguilar-Santamaría et al., 2013). Latex was solubilized in 10% DMSO in PBS by sonication. After the treatment period, 25 µl MTT solution (5 mg/mL) was added, and the plates were incubated for 3 h. After incubation, the supernatant was removed and 200 µL of DMSO was added. Absorbance was measured on a Microplate Reader (Biotek Elx-808) at a wavelength of 630 nm. Cytotoxicity was expressed as cell viability (Abs from treated cell population x 100/Abs from untreated cell population).

2.8 Acute Toxicity The acute oral toxicity of janaguba latex has been assessed following OECD Guideline 423 (OECD, 2001). Three animals per group were used. The experimental group was administered 2,000 mg/kg of H. drasticus latex diluted in 10% DMSO in PBS orally. The other group received the dilution vehicle (10% DMSO in PBS). The

animals were fasted 3 h before administration of each treatment. Following administration the animals were continuously observed for the first 2 h and then every 24 h daily for 14 days. During this period the parameters including food intake, water intake, animal weight and behaviour were evaluated. Then the animals were euthanized, and their organs were collected and submitted to histopathological procedures. 2.9 Histopathological procedures The liver, kidney and spleen of mice were cleaved and immersed in a formaldehyde solution (10% v/v) for 48 h. The fragments were dehydrated in ethyl alcohol in increasing concentrations, xylol diaphanized, impregnated and embedded in paraffin. The blocks were cut in microtome adjusted to 4 µm. The obtained sections were kept at 60 ºC for 24 h and stained with hematoxylin-eosin (HE). The histological images of these slides were captured by a digital camera (Moticam 3000) coupled to the optical microscope (Nikon E-200) under fixed focus and field clarity for liver and kidney slides. Photomicrographs were analyzed using ImageJ software version 1.44 (Research Services Branch, U.S. National Institutes of Health, Bethesda, MD, USA).

2.10 Genotoxicity and mutagenicity The mice were divided into three groups (n = 5). The test group received a 2,000 mg/kg dose of the latex from H. drasticus. The negative control group received the vehicle by gavage (10% DMSO in PBS) and the positive control group received the mutagenic compound cyclophosphamide (25 mg/kg) intraperitoneally (Oliveira et al., 2016). The animals were anesthetized and peripheral blood samples from each animal were collected 48 h after the treatment for the micronucleus and the comet assays (Collins et al., 2008). The experiments were performed in duplicate. After blood collection, the animals were euthanized by cervical dislocation.

2.10.1 Micronucleus Test (MNT) The micronucleus test was performed according to the protocol described by Hayashi et al. (1990). To perform the test 5 µL of blood was dispensed on the middle of a glass slide prepared with acridine orange. Four slides were made from each animal. With the aid of the Zeiss-imager M2 fluorescence microscope and Alexa-fluor-488 nm filter, 2,000 polychromatic erythrocytes were analyzed for the presence of micronuclei (OECD, 2016).

2.10.2 The comet assay The comet assay was performed according to the procedures described by Rocha et al. (2019). A 15 µL blood sample collected from each animal was homogenized with 100 µL low temperature melting agarose. The mixture was then placed on a slide previously coated with standard agarose. Subsequently, the slides were immersed in a lysis solution (2,5 M NaCl, 100 mM EDTA, 10 mM Tris, 10% DMSO, 1% Triton X100, pH 10) for 1 h. After lysis, the slides were incubated for 20 min in buffer solution (1 M NaOH and 200 mM EDTA, pH 13) and electrophoresed for 20 min. Subsequently, they were neutralized (Tris-HCl 0,4 M, pH 7,5) for 15 min and dehydrated in absolute alcohol for 5 min. Ethidium bromide (20 µL in 950 µL distilled water) was used for staining and analyses were performed using a Zeiss-imager M2 fluorescent microscope with the Alexa-fluor-546 nm filter. Two slides were prepared for each animal, and 100 nucleoids (per animal) were verified, where the relationship between tail length and comet head size was simultaneously measured (Collins et al., 2008; Coelho et al., 2018). The analyzed nucleoids were visually classified into one of the following classes: 0 (undamaged), 1 (with little apparent damage), 2 (average damage), 3 (medium damage with longer tail), and 4 (maximum damage), as shown in Figure 1. Therefore, the values obtained for each individual ranged from 0 (100 cells × 0) to 400 (100 cells ×

4) and were used to determine the damage index (DI) per animal. Damage frequency (DF) was also calculated as the percentage of all nuclei with some damage (class 1 to class 4) in relation to the total number of nuclei quantified from class 0 to class 4 (total number) (Collins et al., 2008).

2.11 Statistical Analysis The results of the cytotoxicity were expressed as mean ± SD and analyzed by ANOVA, followed by the Bonferroni test using GraphPad Prism 7.0. For the all the other analyses, Mann-Whitney was performed using SPSS 15.0 (Statistical Package for Social Sciences) software and the data were expressed as mean ± SD. In all tests, p <0.05 were considered statistically significant.

3. Results and discussion Preliminary phytochemical analysis of H. drasticus latex revealed the presence of terpenes, steroids, and their glycosides (Table 1). Luz et al. (2014) reported a similar phytochemical profile of janaguba latex. Saponins are amphiphilic glycosides that have a lipophilic region consisting of a triterpene or steroid moiety and a hydrophilic portion composed of sugars. These compounds reduce the surface tension of water, having detergent, and emulsifier properties. These properties allow them to form complexes with cell membrane components, impacting directly on cell permeability (Schenkel et al., 2001). Studies have shown that saponins have anti-inflammatory, antihelmintic, antiviral, and molluscicidal activities (Silva et al., 2005; Braz Filho, 2010; Costa et al., 2011).

Figure 1. DNA damage classification of cells analyzed by comet assay. 0 (no apparent damage); 1 (with little apparent damage); 2 (average damage); 3 (medium damage with longer tail) and 4 (maximum damage).

Table 1. Secondary metabolites classes found in the latex from H. drasticus. Secondary metabolite classes

Latex of H. drasticus

Flavonoids

-

Phenylpropanoids

-

Triterpenes

+

Steroids

+

Saponins

+++

Monoterpenes and Sesquiterpenes

tr

Alkaloids

-

Couramins

-

Quinones

-

Proanthocyanidins

-

Hydrolysable tannins

-

Reducing sugars

++

Legend: (tr) traces; (-) absent; (+) weak; (++) medium; (+++) strong

In addition to saponins, triterpenes and steroids were identified in the latex. It is noteworthy that there have been previous reports regarding the presence of these isoprenoids in the latex of other species of the genus Himatanthus (Rebouças et al., 2011; Ramos et al., 2015; Soares et al., 2015). For that reason, GC-MS analysis of the nonpolar fraction of the latex was performed, and 16 compounds were identified based on their mass spectra (Stiti and Hartmann, 2012; Almeida et al., 2017), mainly lupeol acetate, botulin, and α/β-amyrin derivatives. Lupeol acetate represented approximately 48.42% of the nonpolar fraction (Table 2). Patocka (2003) and Lucetti et al. (2010), when evaluating the composition of H. drasticus latex, also confirmed the presence of triterpenes such as lupeol acetate. This compound has been shown to be pharmacologically active, with bactericidal and anti-inflammatory activities. A triterpene-rich fraction of H. drasticus latex, composed of lupeol, α-amyrin, and βamyrin, showed promising anti-inflammatory activity through the inhibition of inflammatory enzymes, oxidative stress, and NF-κB (Almeida et al, 2019). Many

studies have shown that the latex of several Himatanthus species contains triterpenes such as lupeol, α-amyrin, β-amyrin, and their acetate and phenylpropanoid esters (Vanderlei et al., 1991; Barreto et al., 1998; Silva et al., 1998; Miranda et al., 2000; Baratto et al., 2010).

Table 2. GC-MS analysis of the nonpolar fraction of H. drasticus latex.

Peak

Retention time (min)

Compounds

Area (%)

1

84.538

Oleanan-3(5),12-diene

1.99

2

85.299

Handianol

1.63

3

90.026

24-Norursa-3,12-diene

2.11

4

90.513

24-Noroleana-3,12-diene

1.34

5

92.174

Lupeol acetate

48.42

6

94.255

Betulin

1.98

7

94.886

β-Amyrin acetate

2.37

8

95.410

Not identified

2.42

9

97.822

3-epi-Betulin

4.62

10

98.205

Not identified

5.72

11

99.570

α-Amyrin acetate

1.68

12

109.854

Fucosterol

1.30

13

112.029

α-Amyrin

4.29

14

115.974

β-amyrone

1.20

15

118.870

Betulinaldehyde acetate

17.70

16

127.432

Not identified

1.23

Total of identified compounds (%)

90.63

The total sugar content of H. drasticus latex was 10.9%, of which 0.94% were reducing sugars. Regarding the protein levels, it was found an amount of 1.2 mg/mL. H. drasticus latex is composed of several secondary metabolites and proteins, which present higher concentrations when compared to the leaves of the species (Agrawal and Konno, 2009; Mithöfer and Boland, 2012).

The effects of H. drasticus latex in Hela, S-180, and EAC cancer cells, and murine peritoneal macrophages showed no cytotoxicity in concentration up to 100 µg/mL, except for S-180 cells, which presented mild reduction in cell viability (approximately 70% of viable cells) when treated with 50 and 100 µg/mL of janaguba latex. This effect was considered as a nonspecific toxicity since the IC50 values were higher than 100 µg/mL (Abdel-Hameed et al., 2012). Santos et al. (2018a) evaluated the antitumor potential of latex in an animal model using S-180 and found that there was a reduction in ascites fluid volume. Mousinho et al. (2011) evaluated the cytotoxicity of janaguba latex proteins against human cancer cell lines HL-60 (leukemia), MDA-MD-435 (melanoma), SF-295 (brain) and HCT-8 (colon) and determined the absence of cytotoxicity for all strains tested, corroborating the results obtained in the present study (Figure 2).

Figure 2. Cell viability of H. drasticus latex in S-180 (sarcoma-180), EAC (Ehrlich ascites carcinoma), Hela (cervical epithelial carcinoma), and MPM (murine peritoneal macrophages) cells. Statistical differences between experimental groups and controls were determined using ANOVA followed by the Bonferroni test. *P > 0.05 vs control. Each value represents the mean ± SD (bars) of three independent experiments.

The evaluation of oral toxicity in mice allows to characterize the potential toxic effects of active components and to estimate the degree of risk of inappropriate consumption (OECD, 2001). Regarding the acute toxicity test, no clinical signs of toxicity and no deaths were observed in animals treated with the 2,000 mg/kg dose of H. drasticus latex. There was no significant difference in water intake between the groups, and there were no significant changes in weight of animals (control: 46.52 ± 0.19 g and latex: 43.83 ± 0.11 g, p > 0.05). Macroscopic analysis did not indicate significant changes in vital organs. There were no signs of ischemia, bleeding, fibrosis or degenerative proliferation in treated mice, as well as no significant difference in relative organ mass (Table 3). Taken together, these results indicate that the latex has no effects in the central nervous system. However, the absence of clinical signs of toxicity and macroscopic analysis is not enough to ensure the safety of an herbal product (Bonomini et al. 2017).

Table 3. Relative mass of organs after euthanasia of H. drasticus latex-treated male mice.

Organs

Control (10% DMSO in PBS)

Latex (2,000 mg/kg)

Spleen (g)

0.249 ± 0.04

0.246 ± 0.09

Heart (g)

0.257 ± 0.06

0.222 ± 0.06

Stomach (g)

0.617 ± 0.09

0.676 ± 0.01

Liver (g)

2.764 ± 0.04

2.932 ± 0.02

Lung (g)

0.461 ± 0.01

0.427 ± 0.02

Right kidney (g)

0.395 ± 0.08

0.385 ± 0.03

Left kidney (g)

0.383 ± 0.07

0.391 ± 0.01

Data are expressed as mean ± SD of n = 3 animals per group. Student T test between groups showed p > 0.05 and were not considered significant.

Histomorphometric analysis of the liver, kidneys, and spleen of mice treated with janaguba latex are shown in Table 4. The data revealed a significant increase in the total number of hepatocytes and Kupffer cells in H. drasticus latex treated animals when compared to the control group. This abnormal proliferation indicates that hepatocytes have been recovering from damage for 14 days (Marinho et al., 2017). The liver is a highly sensitive organ to injury due to its role in xenobiotic metabolism, and synthetic drugs, and even natural substances, can promote liver damage (Singh et al., 2016). The saponins present in the latex may be one cause of the increased number of hepatocytes observed (Li et al, 2016; Wang et al, 2019). The increased number of Kupffer cells may suggest an inflammatory process taking place due to tissue injury in mice treated with janaguba latex. These cells play a crucial role in the phagocytosis of foreign particles and cellular debris, acting to keep the liver function even under stress (Arii and Imanura, 2000).

Table 4. Histomorphometric analysis of the liver, kidney and spleen of mice exposed to H. drasticus latex. Liver (Nº of cells/ µm2) Groups

Latéx

Kidney (areas/µm2)

Spleen (areas/%)

Hepatocyte

Kupffer cell

(%)

(%)

56.81 ± 15.1*

25.28 ± 6.7*

3,733.2 ± 1221.3* 2,793.2 ± 788.9 61.06 ± 6.5

18.86 ± 7.1

3,248.5 ± 1036.9

Control 37.95 ± 11.9

Corpuscle (µm2)

Glomerulus

Stroma

Red pulp

White pulp

(µm2)

(%)

(%)

(%)

2.0 ± 0.48*

36.93 ± 6.49

2,600.8 ± 879.6 60.59 ± 10.8 1.57 ± 0.95

Statistically significant differences were evaluated using Mann-Whitney test with p < 0.05. Data are expressed as the mean ± SD of n = 3 animals per group.

There was also a significant increase in the corpuscular area of the kidneys in the mice administered with H. drasticus latex. It is noteworthy that the kidney is also an organ that is sensitive to toxicity. This is because foreign substances, when metabolized,

37.83 ± 11.14

interact with cellular organelles, generating changes in the cellular signalling pathways. Tissue inflammation and even tissue necrosis can occur as a result (Barnett and Cummings, 2019). Regarding splenic tissue, a significant increase in the area of red pulp was observed. When performing drug treatments in vivo, the spleen often suffers ultrastructural damage that occurs not only by the harmful action of the compounds, but also as a result of the activity of the organism own defence cells (El-Shenawy et al., 2017). However, the histopathological examination of liver tissue showed no sign of damage (cellular edema, steatosis, or necrotic cells). In the kidneys, the glomerular architecture, Bowman's capsule, and renal tubules were well preserved. As for the spleen, the splenic structure also persisted, with well-defined lymph nodes (Figure 3). This is in accordance with results presented by Bonomini et al. (2017), who reported that the ethanolic extract of Allamanda cathartica, belonging to the same family of the species under study (Apocynaceae), did not present systemic toxicity at a dosage of 2,000 mg/kg. Santos and collaborators (2018b) performed the acute toxicity test to evaluate the effects of a H. drasticus popular phytopreparation using a zebrafish model (Danio rerio fish) at concentrations between 20 and 100 µg/mL, and no mortality has been observed. It is noteworthy that the estimated LC50 value was 475 µg/mL. They found mild pathological changes in livers and kidneys of fish treated with concentrations above 750 µg/mL of the phytopreparation. Considering the amount of phytopreparation consumed by people (around 7.14 mg/kg/day), the authors concluded that janaguba milk has low toxicity, but advise to the risks of taking higher doses, especially for cancer patients.

Figure 3. Photomicrographs of different tissues submitted to acute toxicity studies. A (Control) and B (Treated) – Note an increase in the number of hepatocytes in the liver of animals treated with the latex: Hepatocytes (arrows). C (Control) and D (Treated) – See an increase in the renal corpuscle area of animals treated with latex: Glomerulus (asterisk). E (Control) and F (Treated) – Observe an increase in the red pulp area in the spleen of animals treated with latex: RP (red pulp), WP (white pulp). H&E staining, 10X magnification for spleens, and 400x for kidneys and livers.

The genotoxicity test revealed a significant difference between the negative control group and the H. drasticus latex-treated group (Table 5). This result demonstrated that the latex has genotoxic effect, as observed through evaluation using the comet assay (Figure 4). According to Ouedraogo et al. (2012), to obtain authorization and registration of medicinal plants in products from certain countries, genotoxicity testing is required. It is noteworthy that the comet assay is considered one of the most necessary instruments used for the analysis of DNA damage. The assays have a high level of sensitivity and it is applied to a wide spectrum of cells as a standard used to test the safety of new drugs (Tice et al, 2000).

Table 5. Evaluation of the genotoxicity of H. drasticus latex. Groups

DI

DF

Média ± DP

Média ± DP

Negative control

53.4 ± 33.5

28.4 ± 15.2

Positive control

351.0 ± 31.3*

100.0 ± 0.0*

Latex of H. drasticus

129.8 ± 33.4*

71.2 ± 6.8*

Statistically significant differences were evaluated using Mann-Whitney test with *p < 0.05. Data are expressed as the mean ± SD of n = 3 animals per group. DI: Damage index by comet assay; DF: damage factor by comet assay.

The evaluation of mutagenic activity of H. drasticus latex did not revealed any difference between the negative control (0.05 ± 0.03%) and the group treated with the latex (0.16 ± 0.11%). Mice treated with cyclophosphamide showed 33.4 ± 13.9% of micronuclei. Therefore, the treatment cannot be considered a mutation-inducing agent in the tested conditions according to the micronucleus test (Figure 4).

Figure

4.

Fluorescence

microscopy

of

micronucleated

erythrocytes.

Green-stained

normochromatic erythrocytes; Polychromatic red cells stained in red; Micronucleated polychromatic erythrocytes pointed by the white arrow.

From the data obtained, the latex was not considered mutagenic at the dose of 2,000 mg/kg but was genotoxic. On the other hand, Rebolças et al. (2011, 2013) analyzed the in vivo genotoxic and mutagenic effects induced by the latex, aqueous and ethanol extracts from the bark of Himatanthus articulatus, a plant used as an herbal medicine in the Amazon region. The researchers showed that the extracts were not genotoxic, but induced micronucleus formation at the highest dose (2,000 mg/kg). The extracts were still able to reduce clastogenic events, protecting DNA against damage induced by hydrogen peroxide. Latex constituents of plant species can cause irreversible health damage and, consequently, mortality as a result of their mutagenic and genotoxic nature (Verschaeve et al., 2017). Thus, there is a potential interest in the evaluation of mutagenicity and genotoxicity after in vivo exposure to H. drasticus, since this species is widely used in folk medicine. In conclusion, H. drasticus latex showed toxicity signals at high doses (2,000 mg/kg). Although the latex was not mutagenic to mice, it was genotoxic in the Comet assay in our experimental conditions. Even testing a limit dose of 2,000 mg/kg, which is between 10 to 35-fold the amount used in folk medicine, caution must be taken since there is no safe level for genotoxic compounds exposure. Further studies on the toxicological aspects of H. drasticus latex are necessary to elucidate its possible mechanisms of genotoxicity.

Acknowledgments The authors thank to Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE) for the financial support.

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