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Chemotherapeutic and Safety Profile of a Fraction from Mimosa caesalpiniifolia Stem Bark

Chemotherapeutic and Safety Profile of a Fraction from Mimosa caesalpiniifolia Stem Bark Hindawi Journal of Oncology Volume 2021, Article ID 9031975, 12 pages https://doi.org/10.1155/2021/9031975 Research Article Chemotherapeutic and Safety Profile of a Fraction from Mimosa caesalpiniifolia Stem Bark 1,2 1,2 Paulo Michel Pinheiro Ferreira , Renata Rosado Drumond , 1,2 1 Jurandy do Nascimento Silva , Ian Jhemes Oliveira Sousa , 2,3 2,3 Marcus Vinicius Oliveira Barros de Alencar , Ana Maria Oliveira Ferreira da Mata , 4 4 Nayana Bruna Nery Monção , Antonia Maria das Graças Lopes Cito´ , 5 6 7 Ana Fontenele Urano Carvalho , Davi Felipe Farias , Patrı´cia Marçal da Costa , 1 2,3 Adriana Maria Viana Nunes , João Marcelo de Castro e Sousa , 2,3 and Ana Ame´lia de Carvalho Melo-Cavalcante Laboratory of Experimental Cancerology (LabCancer), Department of Biophysics and Physiology, Federal University of Piau´ı, Teresina, Brazil Postgraduate Program in Pharmaceutical Sciences, Federal University of Piau´ı, Teresina, Brazil Laboratory of Genetic Toxicology (Lapgenic), Department of Biochemistry and Pharmacology, Federal University of Piau´ı, Teresina, Brazil Department of Chemistry, Federal University of Piau´ı, Teresina, Brazil Department of Biology, Federal University of Ceara, Fortaleza, Brazil Department of Molecular Biology, Federal University of Para´ıba, João Pessoa, Brazil Faculty of Medicine, State University of Ceara, Fortaleza, Brazil Correspondence should be addressed to Paulo Michel Pinheiro Ferreira; pmpf@ufpi.edu.br Received 23 July 2021; Revised 22 October 2021; Accepted 17 November 2021; Published 7 December 2021 Academic Editor: Liren Qian Copyright © 2021 Paulo Michel Pinheiro Ferreira et al. +is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Mimosa caesalpiniifolia (Fabaceae) is used by Brazilian people to treat hypertension, bronchitis, and skin infections. Herein, we evaluated the antiproliferative action of the dichloromethane fraction from M. caesalpiniifolia (DFMC) stem bark on murine tumor cells and the in vivo toxicogenetic profile. Initially, the cytotoxic activity of DFMC on primary cultures of Sarcoma 180 (S180) cells by Alamar Blue, trypan, and cytokinesis block micronucleus (CBMN) assays was assessed after 72 h of exposure, followed by the treatment of S180-bearing Swiss mice for 7 days, physiological investigations, and DNA/chromosomal damage. DFMC and betulinic acid revealed similar in vitro antiproliferative action on S180 cells and induced a reduction in viable cells, induced a reduction in viable cells and caused the emergence of bridges, buds, and morphological features of apoptosis and necrosis. S180-transplanted mice treated with DFMC (50 and 100 mg/kg/day), a betulinic acid-rich dichloromethane, showed for the first time in vivo tumor growth reduction (64.8 and 80.0%) and poorer peri- and intratumor quantities of vessels. Such antiproliferative action was associated with detectible side effects (loss of weight, reduction of spleen, lymphocytopenia, and neutrophilia and increasing of GOT and micronucleus in bone marrow), but preclinical general anticancer properties of the DFMC were not threatened by toxicological effects, and these biomedical discoveries validate the ethnopharmacological rep- utation of Mimosa species as emerging phytotherapy sources of lead molecules. 2 Journal of Oncology (Teresina, Piauı, Brazil). Air-dried plant material was pul- 1. Introduction verized, extracted with ethanol, concentrated under reduced +e history of anticancer drugs is closely related to natural pressure, and subjected to successive partitioning with products, since at least 60% of clinical drugs naturally or dichloromethane as described by Silva et al. [15]. Previously, chemically resemble ones [1]. In this context, Brazil remains we isolated betulinic acid [3β-hydroxy-lup-20(29)-en-28-oic at the top of 17 megadiverse countries and the home of acid] and verified it as the main compound in the around 20% of the world species [2], mainly because ap- dichloromethane fraction (∼70.3%), as demonstrated by proximately 700 new animal species have been discovered TLC (thin-layer chromatography), GC-qMS (gas chro- each year [3]; it has the greatest number of endemic species matograph quadrupole mass spectrometer), HRAPCIMS on a global scale and about 55,000 plant species (22% of the (high-resolution atmospheric pressure chemical ionization 1 13 world total) [4, 5]. Moreover, it is a great producer of mass spectrometer), H- and C-nuclear magnetic reso- medicinal plants for internal consumption as well as for nance, and DEPT analysis [15, 23]. Plant samplings were international markets. +is invaluable biodiversity encour- authorized by the System of Authorization and Information ages biotechnological and pharmacological studies about on Biodiversity (SISBIO/BAMA #50090-3) and registered in effective therapy and health recovery [6–8]. SisGen (Sistema Nacional de Gestão do Patrimonio ˆ Gen´etico A Brazilian dry region named “Caatinga” dominates 7% e do Conhecimento Tradicional Associado #ABC4AC2) of the Brazilian territory and is an exclusive biome. It according to Brazilian legislation (Federal Law No 13,123/ generates particular environmental conditions for steppe 2015). +ese investigations were performed using the climate-adapted flora and fauna and a high number of rare fraction composed of a mixture of molecules because such and endemic taxa [9, 10], exhibiting many vegetal families, preparations represent the main folk approach of con- such as Fabaceae, Anarcadiaceae, Caryocaraceae, Rhamna- sumption by the Brazilian population [15]. ceae, Chrysobalanaceae, Clusiaceae, Connaraceae, Sapin- daceae, Annonaceae, Combretaceae, and Bignoniaceae 2.2. Animal’s Facilities. Adult female Swiss mice (Mus [11–15]. Among them, inflorescences from Mimosa cae- musculus Linnaeus, 1758) weighing 20–25 g were obtained salpiniifolia Benth. (synonym: Mimosa caesalpiniaefolia, from the animal facilities at Universidade Federal do Piau´ı family Fabaceae), known as “unha de gato,” “sabia,” ´ and (UFPI), Teresina, Brazil. All animals were maintained in sansão-do-campo,” have been traditionally used by Brazilian well-ventilated cages under standard conditions of light people as hedges and windbreaks. Dried fruits and leaves are (12 h with alternate day and night cycles) and temperature given as fodder for cattle, goats, and sheep (crude protein (25± 2 C) with free access to food (Nutrilabor , Campinas, ranging from 13.4 to 17.1%) [16] and to treat hypertension Brazil) and drinkable water. After the tests, mice were eu- [17]. Its bark is popularly used as a coagulant to stop or avoid thanized with sodium thiopental (100 mg/kg) (i.p.). All bleeding and as wound washing to prevent inflammation protocols were approved by the Ethical Committee on and skin infections. Moreover, the ingestion of bark infusion Animal Experimentation at UFPI (CEUA #034/2014) and alleviates symptoms of bronchitis [16, 18, 19]. Recently, a followed Brazilian (Sociedade Brasileira de Ciˆencia em bioassay-guided phytochemical study found 28 compounds Animais de Laboratorio ´ –SBCAL) and international (Di- in M. caesalpiniifolia leaf extract, and four of them revealed rective 2010/63/EU of the European Parliament and of the potent antifungal properties against Candida glabrata and Council on the protection of animals used for scientific Candida krusei [20]; the latter was often associated with the purposes) rules on the care and use of experimental animals. prior use of azoles and corticosteroids, bone marrow transplantation, malignant hematological diseases, and neutropenia [21]. 2.3. In vitro Antiproliferative Studies on Sarcoma 180 Cells An expert Brazilian research group about pharmacology of natural products confirmed bioactivity usages for car- 2.3.1. Ex vivo Cytotoxic Action. Mice with 9 to 10 days of diovascular diseases. +ey reported that ethanolic extracts of S180 ascitic tumors were euthanized with an overdose of different parts of M. caesalpiniifolia (leaves, bark, fruit, and sodium thiopental, and a suspension of S180 cells was taken inflorescences) cause vasorelaxation, the tea of flowers from the intraperitoneal cavity under aseptic conditions. +e promotes hypotension and tachycardia, and the ethanolic cell suspension was centrifuged at 2,000 rpm for 5 min to extract causes hypotension and bradycardia [22]. Based on obtain a pellet, which was washed three times with sterile these ethnopharmacological properties, this work evaluated RPMI medium. +e cell concentration was adjusted to the antiproliferative action of the dichloromethane fraction 0.5 ×10 cells/mL in RPMI-1640 medium supplemented from M. caesalpiniifolia (DFMC) stem bark on murine with 20% fetal bovine serum, 2 mM glutamine, 100 U/mL tumor cells and the in vivo toxicogenetic profile. penicillin, and 100 μg/mL streptomycin (Cultilab , Brazil), plated in 96-well plates with increasing concentrations (0.8–50 μg/mL) of DFMC and betulinic acid, and incubated 2. Materials and Methods at 37 C in a 5% CO atmosphere (Shel Lab CO Incubator, 2 2 2.1. Plant Collection and Extract/Fraction Preparation. USA). Plant specimens were collected in May 2010 in Teresina Cell proliferation was assessed by the Alamar Blue (Piau´ı, Brazil). A voucher sample (26.824) was deposited at assay after 72 h. At 48 h of incubation, 20 μL of stock solution Graziela Barroso Herbarium at Federal University of Piau´ı (0.156 mg/mL) of Alamar Blue (Resazurin, Sigma ™ Journal of Oncology 3 Aldrich , USA) was added to each well. Cell proliferation respective differential percentage and total leukocyte count. was determined spectrophotometrically using a multiplate For biochemical analysis, blood samples were centrifuged at reader (T80+ UV/VIS Spectrometer, PG Instruments , 2,000 rpm for 5 minutes. Physiological markers of the liver United Kingdom) at 570 and 595 nm. +e antiproliferative [blood urea nitrogen (BUN), glutamate oxaloacetate effect was expressed as the percentage of the control transaminase (GOT), glutamate pyruvate transaminase according to Ferreira et al. [12]. (GPT), alkaline phosphatase (ALP)] and kidneys (creati- nine) were evaluated according to Labmax Plenno Labtest . Subsequently, all animals were euthanized to dissect out the 2.3.2. Trypan Blue Exclusion Assay. Sarcoma 180 cells liver, kidneys, spleen, stomach, heart, and lungs to estimate (0.5 ×10 cells/mL) plated in 24-well plates were exposed to wet relative weights and for macroscopic analysis. Next, DFMC at 5, 10, and 25 μg/mL. Doxorubicin (Dox, 0.3 μg/ organs were fixed with 10% buffered formalin, processed, mL) was used as a positive control. Cell viability was ex- and cut into small pieces to prepare histological sections amined by the exclusion of trypan blue [24]. Briefly, aliquots (4–7 μm). Staining was carried out with hematoxylin and of 10 μL were collected from DFMC-treated S180 cultures eosin (H&E, Vetec , Brazil). Morphological blind analyses after 72 h of exposure, and viability was separated into viable were performed under light microscopy (Olympus , Japan) blue-marked and nonviable blue-coloured cells in a Neu- by an expert pathologist. bauer chamber under light microscopy (Biosystems , USA). 2.4.2. Determination of Chromosomal Damages. +e femurs 2.3.3. Cytokinesis-Block Micronucleus (CBMN) Assay. were removed and carefully cleaned, and proximal epiphyses Sarcoma 180 cells were plated in 24-well plates were sectioned. Bone marrow samples were collected using (0.5 ×10 cells/mL) and treated with DFMC at 5, 25, and 5 mL syringes filled with 0.5 mL of sterile fetal bovine serum 50 μg/mL. After 44 h at 37 C, cytochalasin B (Sigma Aldrich, (Cultilab , Brazil), centrifuged, and homogenized. Drops of USA, 6 μg/mL) was added, and the cells were reincubated for cell suspension were transferred to slides to prepare smears an additional 28 h. At 72 h, the cultures were transferred to (two slides/animal), fixed and stained by the Leishman tubes and centrifuged at 800 rpm for 5 minutes. +en, the method. All analyses were blindly performed under light supernatant was removed, and the body of the cell bottom microscopy (Olympus , Japan) with magnifications of 200x was enlarged and centrifuged again before the addition of and 400x. We considered micronuclei to be rounded 2 mL of fixing solution (methanol and acetic acid, ratio 5 :1) structures, with a diameter of 1/5 to 1/20 found in young and 3 drops of formaldehyde 37% (Vetec , Brazil). +is erythrocytes and identified by bluish staining. A total of procedure was repeated 3x using fixing solution 3 :1 without 1,000 polychromatic erythrocytes (PCEs) was quantified per formaldehyde. Supernatants were discarded, and 2-3 drops slide (two slides/animal) [28–30]. of cell suspension were dripped onto slides and stained with Giemsa for 5 min [25]. Considering blind examination, a 2.5. Statistical Analysis. Half maximal inhibitory concen- total of 2000 cells by concentration were counted by optical tration (IC ) and their 95% confidence intervals were microscopy at 1000x (1000 cells/slide) to count buds, calculated by nonlinear regression (GraphPad Prisma 9.0, bridges, and micronuclei. Intuitive Software for Science, USA). Statistical differences were evaluated comparing data [mean± standard error of mean (S.E.M.)] by one-way analysis of variance (ANOVA) 2.4. In vivo Assays and Newman–Keuls test as post hoc test (p< 0.05). All in 2.4.1. Assessment of Antitumor Capacity, Physiological Pa- vitro studies were carried out in duplicate (n � 3/concen- rameters, and Histological Aspects. Ten-day-old S180 ascites tration) and represent independent biological evaluations. tumor cells were removed from the peritoneal cavity, counted (6 ×10 cells/mL) and subcutaneously implanted 3. Results into the right hind axillary of healthy Swiss animals. On the next day, they were randomly divided into four groups 3.1. In vitro Antiproliferative Action on Sarcoma 180 Cells: (n � 10 each). DFMC dissolved in dimethylsulfoxide (DMSO Cytotoxicity, Chromosomal Changes, and Cell Death Pattern. 5%, Vetec , Brazil) was intraperitoneally injected at 50 or DFMC and betulinic acid revealed similar in vitro anti- 100 mg/kg/day for 7 days. Negative and positive controls proliferative activity against S180 cells after 72 h of incu- received DMSO 5% and 5-fluoruracil (5-FU, 25 mg/kg/day, bation, with IC values of 29.0 (24.9–33.6) μg/mL and 33.7 Sigma Aldrich , USA), respectively [26]. (30.1–37.6) μg/mL, respectively (p> 0.05, Table 1). After- All animals were anaesthetized on day 8 with ketamine wards, this action was confirmed by trypan blue assay (90 mg/kg)-xylazine (4.5 mg/kg) for cardiac puncture blood (Figure 1), a direct method to detect cytotoxicity, which collection [27] using sterile tubes and heparinize pipettes to showed that all concentrations of DFMC (5, 25, and determine hematological parameters (erythrocytes, leuko- 50 μg/mL) reduced the number of viable cells (48.2± 7.1, cytes, platelets, hemoglobin, and hematocrit) in peripheral 87.6± 1.4, and 98.7± 0.5%, respectively) when compared to blood samples using an automatic analyzer of hematologic the negative control (p< 0.05). cells (SDH-3 Vet Labtest , Brazil). +e absolute count of Morphological analysis of DFMC-treated Sarcoma 180 white cellular subtypes was calculated as the product of its cells did not show significant micronucleus induction 4 Journal of Oncology Table 1: Cytotoxic activity of the dichloromethane fraction and betulinic acid from Mimosa caesalpiniifolia (DFMC) stem bark on primary culture of sarcoma 180 cells after 72 h of exposure evaluated by alamar blue assay. IC (μg/mL) Sample Sarcoma 180 cells R DFMC 29.0 (24.9–33.6) 0.9278 Betulinic acid 33.7 (30.1–37.6) 0.9292 Doxorubicin 1.9 (1.4–2.4) 0.9801 Data are presented as IC values and 95% confidence intervals. Doxorubicin was used as positive control. Experiments were performed in duplicate. 48.2 ± 7.1% 64.1 ± 4.1% 87.6 ± 1.4% * 98.7 ± 0.5% µg/mL C Dox 5 25 50 DFMC Figure 1: +e cell number of viable cells was determined by trypan blue staining and analyzed by light microscopy after 72 h of exposure to the dichloromethane fraction from Mimosa caesalpiniifolia (DFMC) stem bark. +e percentage of viability reduction in relation to the negative control is described above. +e negative control (C) was treated with the vehicle used to dilute the tested substance. Doxorubicin (Dox, 0.3 μg/mL) was used as a positive control. +e results are expressed as mean ± standard error of measurement (S.E.M.) from two independent experiments. p< 0.05 compared to the control by ANOVA followed by student Newman–Keuls test. (4.5± 0.7, 5.5± 2.1, and 4.5± 2.1 for 5, 25, and 50 μg/mL, were observed, which indicated a reduction in proliferation (Figures 3(e)–3(l)). 5-FU-treated tumors showed larger respectively) in relation to the negative control (3.5± 0.7, p> 0.05, Figure 2(a)). On the other hand, bridges (14.6± 3.9 blood vessels and well vascularized sarcomas, similar to and 27.0± (2) and buds (13.8± 3.3) were observed at 25 and those noted in negative control tumors (Figure 3(e)). On the 50 μg/mL and 50 μg/mL (p< 0.05) when compared to the other hand, DFMC-treated Sarcoma 180 tumors treated with negative control (2.0± 1.4 and 5.5± 3.5), respectively. Such 50 and 100 mg/kg/day exhibited poorer peri- and intratumor chromosomal damage was corroborated by morphological quantities of vessels. In such tumors, vascularization was features of apoptosis (213.0± 73.5 and 337.0± 57.9) and partially restricted to the adipose tissue surrounding the necrosis (162.5± 60.1 and 189.5± 40.3) at 25 and 50 μg/mL tumor (Figures 3(i)–3(j)). (p< 0.05, Figure 2(b)) in the presence of cell rarefaction and vacuolization. As expected, Dox increased buds (15.5± 3.5) 3.3. Physiological Parameters. In the next step, we assessed and micronuclei (18.5± 4.9) and caused typical findings of macroscopic and microscopic parameters of key organs and apoptosis (466.0± 101.8) and necrosis (177.5± 3.5) (p< 0.05). the hematological profile of Sarcoma 180-bearing mice after treatment with DFMC. First, we found a reduction in body 3.2. In vivo Antitumoral Activity. Experimentally trans- weight gain in DFMC-treated animals (20.6± 0.8 and planted mice with Sarcoma 180 cells and treated with DFMC 21.4± 1.6 g, for 50 and 1000 mg/kg/day) in a similar way to (50 and 100 mg/kg/day) for 7 days revealed a significant the 5-FU group (20.1± 0.9 g) when compared to the negative control (26.3± 2.2 g, p< 0.05, Table 1). Wet relative weight reduction in tumor growth [(0.28± 0.04 g (64.8± 5.3%) and 0.16± 0.07 g (80.0± 8.4%)] when compared to the negative reduction of spleens was noted in both doses of DFMC (0.2± 0.08 and 0.2± 0.03 g/100 g of body weight) and in 5- control (0.80± 0.13 g, respectively). Tumor reduction was also noted in the positive control group treated with 5-FU FU-treated animals (0.2± 0.04 g), but liver decrease was [0.11± 0.03 g (82.8± 4.2%)] (p< 0.05, Table 2). observed in 5-FU-treated animals only (4.7± 0.1 g) in +e negative control group showed characteristics of comparison with the negative group (0.4± 0.04 g and malignant neoplasms consisting of round and polyhedral 6.0± 0.4 g, respectively, p< 0.05). cells, anisocariosis, binucleation, mitoses, and different Hematological analysis of DFMC-treated animals showed degrees of cell and nuclear pleomorphism, chromatin neutrophilia (33.8± 3.2%), lymphocytopenia (61.5± 3.6%), a condensation, and extensive areas of muscle invasion reduction in eosinophils (0.4± 0.2%), and a slight increase in (Figures 3(a)–3(d)). Tumor samples from 5-FU 25 mg/kg/ GOT levels (315.3± 8.9 U/mL) (p< 0.05, Table 3). Animals exposed to 5-FU showed intense leukopenia (1.6± 0.3/mm day and FDCM 50 and 100 mg/kg/day also revealed the ) typical morphology of neoplastic cells, although rare mitoses due to declines in neutrophils (12.9± 1.3%), monocytes Cell number (×10 /mL) Journal of Oncology 5 20 * C Dox 5 25 50 µg/mL DFMC Buds Micronucleus Bridges (a) 200 * C Dox 5 25 50 µg/mL DFMC Apoptosis Necrosis (b) Figure 2: Ex vivo chromosomal changes and cell death pattern in sarcoma 180 cells determined by micronucleus assay with cytokinesis block after 72 h exposure to the dichloromethane fraction from Mimosa caesalpiniifolia (DFMC) stem bark. +e negative control (C) was treated with the vehicle used to dilute the tested substance (DMSO 0.1%). Doxorubicin (Dox, 0.3 μg/mL) was used as a positive control. +e results are expressed as mean± standard error of measurement (S.E.M.) from two independent experiments. p< 0.05 compared to the control by ANOVA followed by student Newman–Keuls test. Table 2: Effect of the dichloromethane fraction from Mimosa caesalpiniifolia (DFMC) stem bark on the relative weight of key organs and on the tumor growth of sarcoma 180-bearing swiss mice after 7 days of intraperitoneal treatment. Dose (mg/kg/ Mice weight Liver Kidney Spleen Stomach Lungs Tumor inhibition Substance Tumor (g) day) (g) g/100 g body weight (%) Negative — 26.3± 2.2 6.0± 0.4 1.1± 0.1 0.4± 0.04 1.0± 0.1 0.8± 0.1 0.80± 0.13 — control ∗ ∗ ∗ ∗ ∗ 5-FU 25 20.1± 0.9 4.7± 0.1 1.2± 0.1 0.2± 0.04 1.1± 0.1 0.8± 0.1 0.11± 0.03 82.8± 4.2 ∗ ∗ ∗ ∗ 50 20.6± 0.8 5.8± 0.2 1.2± 0.1 0.2± 0.08 1.1± 0.1 1.0± 0.2 0.28± 0.04 64.8± 5.3 DFMC ∗ ∗ ∗ ∗ 100 21.4± 1.0 5.9± 0.2 1.3± 0.1 0.2± 0.03 1.2± 0.5 0.8± 0.1 0.16± 0.07 80.0± 8.4 Values are means± S.E.M. (n � 10 animals/group). +e negative control was treated with the vehicle used to dilute the drug (DMSO 5%). 5-Fluorouracil (5-FU) was used as positive control. p< 0.05 compared with the negative control by ANOVA followed by Newman–Keuls test. (0.6± 0.2%) and eosinophils (0.6± 0.3%) compared to the from these groups. Livers did not exhibit hyperplasia, he- animals from the negative group (5.1± 0.4/mm , 18.8± 2.8%, mosiderin pigments, infiltration of leukocytes, cell swelling, 1.8± 0.3% and 1.8± 0.4%, respectively, p< 0.05). portal congestion, or areas of necrosis, although micro- esteatosis was detected in all groups (Figure 4(a)). Kidneys present no swelling, tubular degeneration, vascular congestion, 3.4. Histological Alterations. Animals from the negative or necrosis focus (Figure 4(b)); in hearts, there were no areas of control group and treated with DFMC (50 and 100 mg/kg/day) degeneration or fibroblasts proliferation and striations were did not show signs of toxicity, with similarity among organs clearly visible (Figure 4(c)); lungs showed bronchioles and Events (2000 cells) Events (2000 cells) 6 Journal of Oncology AB C D EF G IJ K L Muscular invasion Chromation condensation Intratumoral vessels Binucleation Mitosis Microenvironment vessels Figure 3: Morphology of sarcoma 180 tumor cells from swiss mice after 7 days of treatment with dichloromethane fraction from Mimosa caesalpiniifolia stem bark. Animals were treated by intraperitoneal injection (50 mg/kg/day: g, h, and i; 100 mg/kg/day: j, k, and l). +e negative control was treated with the vehicle used to dilute the substance (DMSO 5%: a–d). 5-Fluorouracil was used as a positive control (e and f). Hematoxylin-eosin staining. Light microscopy magnification, 100x-400x. Table 3: Hematological and biochemical parameters of mice intraperitoneally treated with dichloromethane fraction from Mimosa caesalpiniifolia stem bark for 7 days. Dichloromethane fraction from Mimosa caesalpiniifolia Parameters Negative control 5-FU 25 mg/kg/day 50 mg/kg/day 100 mg/kg/day Erythrocytes (mm ) 4.5± 0.2 4.4± 0.2 5.0± 0.1 4.9± 0.2 Hemoglobin (g/dL) 13.6± 0.7 13.3± 0.8 15.4± 0.4 15.0± 0.7 Hematocrit (%) 40.7± 2.3 40.1± 2.3 46.3± 1.1 44.9± 2.2 VCM (fL) 90.8± 0.5 90.6± 0.6 91.8± 0.3 91.6± 0.5 HCM (pg) 30.2± 0.2 30.2± 0.2 30.5± 0.1 30.5± 0.2 CHCM (g/dL) 33.3± 0.1 33.1± 0.1 33.2± 0.1 33.3± 0.1 Platelets (mm ) 3.6± 0.5 2.9± 0.2 3.4± 0.2 3.4± 0.2 3 ∗ Total leukocytes (mm ) 5.1± 0.4 1.6± 0.3 5.4± 0.6 4.9± 0.7 ∗ ∗ Neutrophils (%) 18.8± 2.8 12.9± 1.3 23.3± 4.1 33.8± 3.2 Rods (%) 0.4± 0.2 0.4± 0.3 0.6± 0.2 1.8± 0.7 Lymphocytes (%) 77.3± 3.0 85.6± 1.8 73.7± 4.1 61.5± 3.6 Monocytes (%) 1.8± 0.3 0.6± 0.2 1.7± 0.6 2.6± 0.8 ∗ ∗ ∗ Eosinophils (%) 1.8± 0.4 0.6± 0.3 0.7± 0.3 0.4± 0.2 Basophils (%) 0.0 0.0 0.0 0.0 GOT (U/mL) 286.9± 5.8 303.2± 7.6 280.8± 9.1 315.3± 8.9 GTP (U/mL) 158.8± 2.6 157.5± 4.3 161.6± 5.0 156.3± 1.1 ALP (U/L) 112.3± 5.5 131.2± 9.8 101.2± 3.4 93.8± 6.6 Creatinine (mg/dL) 0.5± 0.05 0.5± 0.08 0.4± 0.01 0.4± 0.04 BUN (mg/dL) 48.9± 4.2 37.7± 2.4 41.3± 6.6 42.8± 3.1 MCH, mean corpuscular hemoglobin; MCV, mean corpuscular volume; MCHC, mean corpuscular hemoglobin concentration; BUN, blood urea nitrogen; GOT, glutamate oxaloacetate transaminase; GPT, glutamate pyruvate transaminase; ALP, alkaline phosphatase. Values are means± S.E.M. (n � 10 ani- mals/group). +e negative control was treated with the vehicle used to dilute the drug (DMSO 5%). 5-Fluorouracil (5-FU) was used as positive control. P< 0.05 compared with the negative control by ANOVA followed by Newman–Keuls test. visible alveolar spaces, absence of mono and polymorpho- hemorrhagic streaks, a cardiac region with a keratinized nuclear cells or areas of necrosis (Figure 4(d)); stomachs squamous lining, no changes in chorion and easy visualization showed normal mucosa and submucosa, absence of of parietal and main cells (Figure 4(e)). Spleens showed Journal of Oncology 7 Kuper cell ff Tubular space Centrilobular vein Capsular space Glomerulus Arterioles Liver Duct Microesteatosis Proximal tubule (a) (b) Normal fibrocytes Pneumocyte type II Transverse streaks Pneumocyte type I Intercalated discs Capillary Nucleus Alveolar sac (c) (d) Muscus cells Gastric glands Gastric pits (e) Figure 4: General morphology of livers (a), kidneys (b), hearts (c), lungs (d), and stomachs (e) from Swiss mice after 7 days of treatment with dichloromethane fraction from Mimosa caesalpiniifolia stem bark (50 or 100 mg/kg/day) or vehicle used to dilute the substance (DMSO 5%). Important changes among these groups were not observed. Hematoxylin-eosin staining. Light microscopy magnification, 400x. megakaryocytes and hemosiderin pigments in all groups. indisputable advantage when compared to traditional Disorganization of lymphoid follicles and relative reduction of treatments based on surgery and monochemotherapy, the white pulp were observed in the 5-FU (Figure 5(b)) and making it possible to cure neoplasms such as acute child- DFMC-treated animals (Figures 5(c) and 5(d)). On the other hood leukemia, Hodgkin and non-Hodgkin’s lymphomas, hand, 5-FU-treated animals showed slight hepatocyte swelling and germ cell tumors [31, 32]. However, the great hetero- and suggestion of mild changes in fatty metabolism since geneity of tumor cells makes treatment difficult and facili- macroesteatosis was noted, and kidneys presented swelling of tates the manifestation of resistance [33], which stimulates tubular cells and foci of atrophic glomeruli (results not shown). the search for new chemotherapeutic agents. Initially, the antiproliferative action of DFMC was eval- uated in primary cultures of Sarcoma 180 cells. In vitro cy- 3.5. In vivo Chromosomal Damage. DFMC increased totoxicity tests in cell cultures are important for the evaluation micronucleated polychromatic erythrocytes in the bone of antitumor agents, and at least during the screening phase, marrow of mice in a dose-dependent manner (50 and they have reduced in vivo tests on animals. In addition, they 100 mg/kg/day: 11.5± 0.2 and 26.0± 2.1, respectively) are widely used as alternative methods to pharmacological compared to the vehicle group (2.8± 0.2, p< 0.05). As ex- tests on isolated organs [26, 34]. Herein, DFMC and its pected, 25 mg/kg/day 5-FU caused clastogenic effects majority compound betulinic acid revealed similar cytotoxic (14.0± 0.1, p< 0.05). capacity on S180 cells by Alamar blue assay, whose action was confirmed by cell viability reduction in trypan blue exclusion tests. Some reports, including the American National Cancer 4. Discussion Institute (NCI-USA) [35], suggest that IC values around In the last century, the development of cytotoxic agents has 30 μg/mL are a suitable outcome to consider extracts and revolutionized anticancer therapy. Adjuvant treatments with fractions promising substances for further purification and antiproliferative substances have demonstrated an biological studies [12, 15]. Recently, we reported that DFMC 8 Journal of Oncology White pulp White pulp Hemosiderin pigments Megakaryocytes Megakaryocytes Hemosiderin pigments Trabeculae Red pulp (a) (b) Central arteriole Disorganization of follicles Megakaryocytes Megakaryocyte Hemosiderin pigments Disorganization of follicles (c) (d) Figure 5: Spleen morphology of Swiss mice after 7 days of treatment with dichloromethane fraction from Mimosa caesalpiniifolia stem bark (50 mg/kg/day (c); 100 mg/kg/day (d)), vehicle used to dilute the substance DMSO 5% (a) or 5-fluorouracil 25 mg/kg/day (b). Hematoxylin- eosin staining. Light microscopy magnification, 400x. has higher cytotoxic action against different types of tumor block micronucleus (CBMN) assays were performed to tissues (promyelocytic leukemia, HL-60; glioblastoma, SF- measure micronuclei quantification and DNA damage in 295; ovarian, OVCAR-8; colon, HCT-116) than hexane and mammalian cell cultures [28]. Apart from the evaluation of water extracts. DFMC did not produce hemolysis and showed micronuclei, the CBMN cytome assay allows the assessment higher potential as a cytotoxic agent than betulinic acid for the of other relevant biodosimetric markers: nucleoplasmic SF-295 and HL-60 lines [20, 36], corroborating the findings bridges, nuclear buds, proportion of dividing cells (pa- rameter of cytostasis), and cells undergoing apoptosis and described here for S180 cells. Phytochemical investigation of extracts from Mimosa necrosis (parameters of cytotoxicity). +erefore, this tech- nique was updated to detect chromosomal breaks, DNA species revealed the existence of terpenes, flavonoids, ste- roids, phenols (especially tannins), and fatty acid derivatives rearrangements, chromosomal losses, cytostasis, and to in different parts of the plant (leaves, fruits, flowers, separate types of cell death [25, 28, 51, 52]. +erefore, for the branches, and stem bark) [36–40], mainly betulinic acid, first time, an increase in chromosomal damage represented lupeol, phytol, lactic acid, α-tocopherol, stigmasterol, β-si- by (i) nucleoplasm bridges: a biomarker of dicentric chro- tosterol, sitostenone, and stigmasta-4,22-dien-3-one, which mosomes, resulting from the fusion of the final telomeres had been identified in dichloromethane, ethanolic, and after DNA double-strand breaks or DNA misrepair/rear- hexane fractions of leaves and barks from M. caesalpiniifolia rangements; (ii) buds: a biomarker of gene amplification and [15, 36, 40], suggesting that the antiproliferative potential of originating from interstitial or terminal acentric fragments; and (iii) morphological features of apoptosis and necrosis in DFMC may be attributed, at least in part, to its chemo- preventive action. In this context, Silva et al. [15] stated the S180 cells at higher concentrations of DFMC was noted. Meanwhile, both doses of DFMC also induced the emer- scavenger activity of M. caesalpiniifolia extracts, whose presence of phenolic compounds was confirmed by ultra- gence of micronucleated polychromatic erythrocytes in bone violet-visible spectroscopy and thin-layer chromatography. marrow. Previously, Silva et al. [23] reported an ethanolic Betulinic acid, a naturally occurring pentacyclic tri- extract from M. caesalpiniifolia leaves with maximum cy- terpenoid, is the main compound in the fraction (∼70.3%) totoxicity on breast carcinoma MCF-7 cells at 320 μg/mL [15, 23], and both samples (DFMC and isolated molecule) and morphological changes suggestive of apoptosis, in- have similar bioactivity on S180 cells (p> 0.05), confirming cluding DNA fragmentation and nuclear chromatin reports about the antiproliferative action of betulinic acid in condensation. many types of cancers [41–50]. Recently, we also showed that micronuclei formation and changes indicating mutagenic index in DFMC-treated To complement the ex vivo cytotoxic analysis on S180 tumor cells and in vivo pharmacological safety, cytokinesis- roots were not detected, although this fraction has inhibited Journal of Oncology 9 also essential data about the pharmacokinetics profile, growth of Allium cepa roots and increase amount of bridges in dividing meristematic cells, which indicates capacity for therapeutic window, and pharmacological safety, including systemic and genetic toxicology [58, 59]. +ese assessments mitotic index reduction as seen as dropping of cells at metaphase, anaphase, and telophase phases and cycle arrest allow the exclusion of undesirable drug candidates and save at prophase [15]. Regardless, it is likely that DNA/chro- time, material and human resources. In the case of plant mosomal damage is a sign of nonselective mechanism(s) in toxicity/poisoning, its harmful action must be proven ex- tumor or normal dividing cells. +erefore, in vitro (bridges perimentally. For humans, this experimental reproduction and buds) and in vivo (micronucleus) clastogenic findings should be carried out in the same animal species, naturally led to cell cycle arrest as a “cellular escape” from death, affected, or related species (e.g., mice and rats), although different susceptibilities to the effects of toxic herbals among mainly if we consider the antiproliferative action of DFMC on human normal leukocytes well [15]. species are a common mark [60, 61]. Acute signs of systemic toxicity are loss of body mass and Indeed, antineoplastic agents induce DNA strand breaks in mammalian cells, as seen with inhibitors of topoisomerase expansion or involution of key organs in mammals exposed to an investigational drug [62]. Weight loss is one of the I (camptothecin) and topoisomerase II (etoposide) [53] and 5-FU. 5-FU is a widely used antimetabolite to treat breast most common side effects after chemotherapy cycles with 5- adenocarcinomas and cancers of the gastrointestinal tract FU or doxorubicin, since the gastrointestinal system is one and head and neck due to its inhibitory action on the enzyme of the main nonspecific targets of nontarget antiproliferative thymidylate synthase [54], among other mechanisms, de- agents, causing seasickness, suppression of appetite, vom- spite its unblemished in vivo clastogenic activity [55]. iting, and diarrhea [63]. Loss of body weight and reduction However, genotoxicity does not mean mutagenicity because of spleens were macroscopic manifestations found in the 5- FU- and DFMC-treated groups, but signs of diarrhea were some genome injuries are biochemically fixed, which indi- cates that antineoplastic acute toxic consequences (e.g., not seen in the DFMC-treated groups. Spleen diminution is another very common side effect found in S180-bearing inhibition of growth and cell division) are not automatically linked to chromosomal loss/impairments [56]. mice under experimental treatment with promising anti- tumoral substances [26, 64] and reflects lymphocytopenia +e cytotoxic activity on cancer cells using in vitro models may not reflect in vivo findings, since the latter seen in 5-FU- and DFMC-treated groups and strong leu- considers pharmacokinetic and pharmacodynamic vari- kopenia in 5-FU-treated mice, which was confirmed by ables, such as ligand binding to specific receptors, down- disorganization of lymphoid follicles and size reduction of stream cascade, involvement of second messengers, water/ white pulps. lipid solubility, bioavailability, first-pass metabolism, and In vivo toxicological studies with DFMC were not found in renal excretion [57, 58]. +erefore, combining these two the literature, but oral subacute treatment of rats for 32 days with 750 mg/kg/day ethanolic extract from M. caesalpiniifolia types of scientific tools is appropriate for a more complete assessment of a substance with antiproliferative action. For leaves caused weight loss, hepatomegaly, and an increase in adrenal and pituitary glands [40], but serum biochemical the first time, the amazing antitumor action of a dichloromethane fraction from M. caesalpiniifolia stem bark evaluation (alkaline phosphatase, GOT, urea, and creatinine) on in vivo proliferating Sarcoma 180 cells was demonstrated. did not identify renal or liver changes. On the other hand, we In vivo studies have already shown that betulinic acid in- noted that the 100 mg/kg/day DFMC-treated group revealed a hibits the growth of human ovarian IGROV-1 carcinoma slight but significant increase in GOT. xenographic tumors at 100 mg/kg/day and increases the Transaminases (GOTand GTP) are found in all human survival rate of mice [46]. systems and many organs, but they are more present in the No specific changes were noted during histopathological cytoplasm (100% GTP/20% GOT) or mitochondria (80% analysis of the Sarcoma 180 tumors [34], but it is important GOT) of hepatocytes, since they catalyze transamination reactions, working central providers of secondary me- to emphasize that local vascularization from DFMC-treated animals was predominantly confined to the adipose tissue tabolites to the citric acid cycle. Any type of liver injury may sensibly increase serum GTP concentrations, a classic surrounding the tumors. +ese unexpected findings were not described before and suggest that the fraction interferes biomarker to assess acute or chronic hepatic damage, but with the cell cycle of Sarcoma 180 cells and inhibits an- its origin can have kidney, heart, or muscle reasons be- giogenesis, which obviously alters the stromal environment, cause these organs also possess higher GTP concentra- such as the local pH, partial pressure of oxygen and carbon tions in comparison with other tissues [65]. On the other dioxide, input of nutrients/growth factors, and cleaning of hand, GOT is more abundant in heart, skeletal muscle, metabolic residues [57], all essential primary conditions for kidneys, brain, and red blood cells [66], with lower concentrations in skeletal muscle and kidney. Although cellular division and tumor growth. Molecular studies are underway to confirm such antiangiogenic potential. +ese GTP is more specific for detecting liver damage, ischemic or toxic damage to zone 3 of the hepatic acinus may data corroborate our findings about the biomedical potential of M. caesalpiniifolia and inspired us to assess the phar- change GOT levels since this region has greater GOT concentrations [65]. macological safety profile of the fraction, taking into con- sideration its promising phytotherapy properties. Histological changes were not found in livers from +e development of new (phyto)pharmaceutical prod- DFMC-treated animals. +us, it is probable that higher ucts includes not only pharmacodynamic discoveries but levels of GOT may be associated with muscle damage and/ 10 Journal of Oncology In vitro antiproliferative and clastogenic effects upon S180 cells Betulinic acid-rich Slight physiological changes: fraction from Mimosa loss of weight, reduction of spleen, CO H lymphocytopenia and neutrophilia, caesalpiniifolia stem bark GOT increasing, MN in bone marrow HO In vivo tumor reduction ranging from 64.8 to 80 % Figure 6: Summary of the antiproliferative, genotoxic, antitumoral, and toxicological effects of a betulinic acid-rich fraction from Mimosa caesalpiniifolia stem bark. or trauma after continual intraperitoneal injections be- Conflicts of Interest cause this procedure can result in aminotransferase re- +e authors declare that there are no conflicts of interest lease, and an increase in GOT is common in such regarding the publication of this paper. situations [66]. +e majority of clinically available anticancer medica- tions provoke strong side effects, especially suppression of Acknowledgments bone marrow and immune response, toxicity on hepato- cytes, cardiac myocytes and enterocytes, mucositis, weight Paulo Michel Pinheiro Ferreira is grateful to the public and hair loss (incidence of 65%), opportunistic infections, Brazilian agency “Conselho Nacional de Desenvolvimento seasickness, vomiting, chemotherapy-related anorexia, pe- Cient´ıfico e Tecnologico” ´ [CNPq (#303247/2019-3)] for his ripheral neuropatia, and tiredness [33, 63, 67–69], whose personal scholarship. +e authors also thank Leane Brunelle types and intensity depend on the mechanism(s) of action dos Santos Alves for technical assistance and the Post- and idiosyncratic reactions. 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Chemotherapeutic and Safety Profile of a Fraction from Mimosa caesalpiniifolia Stem Bark

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Hindawi Journal of Oncology Volume 2021, Article ID 9031975, 12 pages https://doi.org/10.1155/2021/9031975 Research Article Chemotherapeutic and Safety Profile of a Fraction from Mimosa caesalpiniifolia Stem Bark 1,2 1,2 Paulo Michel Pinheiro Ferreira , Renata Rosado Drumond , 1,2 1 Jurandy do Nascimento Silva , Ian Jhemes Oliveira Sousa , 2,3 2,3 Marcus Vinicius Oliveira Barros de Alencar , Ana Maria Oliveira Ferreira da Mata , 4 4 Nayana Bruna Nery Monção , Antonia Maria das Graças Lopes Cito´ , 5 6 7 Ana Fontenele Urano Carvalho , Davi Felipe Farias , Patrı´cia Marçal da Costa , 1 2,3 Adriana Maria Viana Nunes , João Marcelo de Castro e Sousa , 2,3 and Ana Ame´lia de Carvalho Melo-Cavalcante Laboratory of Experimental Cancerology (LabCancer), Department of Biophysics and Physiology, Federal University of Piau´ı, Teresina, Brazil Postgraduate Program in Pharmaceutical Sciences, Federal University of Piau´ı, Teresina, Brazil Laboratory of Genetic Toxicology (Lapgenic), Department of Biochemistry and Pharmacology, Federal University of Piau´ı, Teresina, Brazil Department of Chemistry, Federal University of Piau´ı, Teresina, Brazil Department of Biology, Federal University of Ceara, Fortaleza, Brazil Department of Molecular Biology, Federal University of Para´ıba, João Pessoa, Brazil Faculty of Medicine, State University of Ceara, Fortaleza, Brazil Correspondence should be addressed to Paulo Michel Pinheiro Ferreira; pmpf@ufpi.edu.br Received 23 July 2021; Revised 22 October 2021; Accepted 17 November 2021; Published 7 December 2021 Academic Editor: Liren Qian Copyright © 2021 Paulo Michel Pinheiro Ferreira et al. +is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Mimosa caesalpiniifolia (Fabaceae) is used by Brazilian people to treat hypertension, bronchitis, and skin infections. Herein, we evaluated the antiproliferative action of the dichloromethane fraction from M. caesalpiniifolia (DFMC) stem bark on murine tumor cells and the in vivo toxicogenetic profile. Initially, the cytotoxic activity of DFMC on primary cultures of Sarcoma 180 (S180) cells by Alamar Blue, trypan, and cytokinesis block micronucleus (CBMN) assays was assessed after 72 h of exposure, followed by the treatment of S180-bearing Swiss mice for 7 days, physiological investigations, and DNA/chromosomal damage. DFMC and betulinic acid revealed similar in vitro antiproliferative action on S180 cells and induced a reduction in viable cells, induced a reduction in viable cells and caused the emergence of bridges, buds, and morphological features of apoptosis and necrosis. S180-transplanted mice treated with DFMC (50 and 100 mg/kg/day), a betulinic acid-rich dichloromethane, showed for the first time in vivo tumor growth reduction (64.8 and 80.0%) and poorer peri- and intratumor quantities of vessels. Such antiproliferative action was associated with detectible side effects (loss of weight, reduction of spleen, lymphocytopenia, and neutrophilia and increasing of GOT and micronucleus in bone marrow), but preclinical general anticancer properties of the DFMC were not threatened by toxicological effects, and these biomedical discoveries validate the ethnopharmacological rep- utation of Mimosa species as emerging phytotherapy sources of lead molecules. 2 Journal of Oncology (Teresina, Piauı, Brazil). Air-dried plant material was pul- 1. Introduction verized, extracted with ethanol, concentrated under reduced +e history of anticancer drugs is closely related to natural pressure, and subjected to successive partitioning with products, since at least 60% of clinical drugs naturally or dichloromethane as described by Silva et al. [15]. Previously, chemically resemble ones [1]. In this context, Brazil remains we isolated betulinic acid [3β-hydroxy-lup-20(29)-en-28-oic at the top of 17 megadiverse countries and the home of acid] and verified it as the main compound in the around 20% of the world species [2], mainly because ap- dichloromethane fraction (∼70.3%), as demonstrated by proximately 700 new animal species have been discovered TLC (thin-layer chromatography), GC-qMS (gas chro- each year [3]; it has the greatest number of endemic species matograph quadrupole mass spectrometer), HRAPCIMS on a global scale and about 55,000 plant species (22% of the (high-resolution atmospheric pressure chemical ionization 1 13 world total) [4, 5]. Moreover, it is a great producer of mass spectrometer), H- and C-nuclear magnetic reso- medicinal plants for internal consumption as well as for nance, and DEPT analysis [15, 23]. Plant samplings were international markets. +is invaluable biodiversity encour- authorized by the System of Authorization and Information ages biotechnological and pharmacological studies about on Biodiversity (SISBIO/BAMA #50090-3) and registered in effective therapy and health recovery [6–8]. SisGen (Sistema Nacional de Gestão do Patrimonio ˆ Gen´etico A Brazilian dry region named “Caatinga” dominates 7% e do Conhecimento Tradicional Associado #ABC4AC2) of the Brazilian territory and is an exclusive biome. It according to Brazilian legislation (Federal Law No 13,123/ generates particular environmental conditions for steppe 2015). +ese investigations were performed using the climate-adapted flora and fauna and a high number of rare fraction composed of a mixture of molecules because such and endemic taxa [9, 10], exhibiting many vegetal families, preparations represent the main folk approach of con- such as Fabaceae, Anarcadiaceae, Caryocaraceae, Rhamna- sumption by the Brazilian population [15]. ceae, Chrysobalanaceae, Clusiaceae, Connaraceae, Sapin- daceae, Annonaceae, Combretaceae, and Bignoniaceae 2.2. Animal’s Facilities. Adult female Swiss mice (Mus [11–15]. Among them, inflorescences from Mimosa cae- musculus Linnaeus, 1758) weighing 20–25 g were obtained salpiniifolia Benth. (synonym: Mimosa caesalpiniaefolia, from the animal facilities at Universidade Federal do Piau´ı family Fabaceae), known as “unha de gato,” “sabia,” ´ and (UFPI), Teresina, Brazil. All animals were maintained in sansão-do-campo,” have been traditionally used by Brazilian well-ventilated cages under standard conditions of light people as hedges and windbreaks. Dried fruits and leaves are (12 h with alternate day and night cycles) and temperature given as fodder for cattle, goats, and sheep (crude protein (25± 2 C) with free access to food (Nutrilabor , Campinas, ranging from 13.4 to 17.1%) [16] and to treat hypertension Brazil) and drinkable water. After the tests, mice were eu- [17]. Its bark is popularly used as a coagulant to stop or avoid thanized with sodium thiopental (100 mg/kg) (i.p.). All bleeding and as wound washing to prevent inflammation protocols were approved by the Ethical Committee on and skin infections. Moreover, the ingestion of bark infusion Animal Experimentation at UFPI (CEUA #034/2014) and alleviates symptoms of bronchitis [16, 18, 19]. Recently, a followed Brazilian (Sociedade Brasileira de Ciˆencia em bioassay-guided phytochemical study found 28 compounds Animais de Laboratorio ´ –SBCAL) and international (Di- in M. caesalpiniifolia leaf extract, and four of them revealed rective 2010/63/EU of the European Parliament and of the potent antifungal properties against Candida glabrata and Council on the protection of animals used for scientific Candida krusei [20]; the latter was often associated with the purposes) rules on the care and use of experimental animals. prior use of azoles and corticosteroids, bone marrow transplantation, malignant hematological diseases, and neutropenia [21]. 2.3. In vitro Antiproliferative Studies on Sarcoma 180 Cells An expert Brazilian research group about pharmacology of natural products confirmed bioactivity usages for car- 2.3.1. Ex vivo Cytotoxic Action. Mice with 9 to 10 days of diovascular diseases. +ey reported that ethanolic extracts of S180 ascitic tumors were euthanized with an overdose of different parts of M. caesalpiniifolia (leaves, bark, fruit, and sodium thiopental, and a suspension of S180 cells was taken inflorescences) cause vasorelaxation, the tea of flowers from the intraperitoneal cavity under aseptic conditions. +e promotes hypotension and tachycardia, and the ethanolic cell suspension was centrifuged at 2,000 rpm for 5 min to extract causes hypotension and bradycardia [22]. Based on obtain a pellet, which was washed three times with sterile these ethnopharmacological properties, this work evaluated RPMI medium. +e cell concentration was adjusted to the antiproliferative action of the dichloromethane fraction 0.5 ×10 cells/mL in RPMI-1640 medium supplemented from M. caesalpiniifolia (DFMC) stem bark on murine with 20% fetal bovine serum, 2 mM glutamine, 100 U/mL tumor cells and the in vivo toxicogenetic profile. penicillin, and 100 μg/mL streptomycin (Cultilab , Brazil), plated in 96-well plates with increasing concentrations (0.8–50 μg/mL) of DFMC and betulinic acid, and incubated 2. Materials and Methods at 37 C in a 5% CO atmosphere (Shel Lab CO Incubator, 2 2 2.1. Plant Collection and Extract/Fraction Preparation. USA). Plant specimens were collected in May 2010 in Teresina Cell proliferation was assessed by the Alamar Blue (Piau´ı, Brazil). A voucher sample (26.824) was deposited at assay after 72 h. At 48 h of incubation, 20 μL of stock solution Graziela Barroso Herbarium at Federal University of Piau´ı (0.156 mg/mL) of Alamar Blue (Resazurin, Sigma ™ Journal of Oncology 3 Aldrich , USA) was added to each well. Cell proliferation respective differential percentage and total leukocyte count. was determined spectrophotometrically using a multiplate For biochemical analysis, blood samples were centrifuged at reader (T80+ UV/VIS Spectrometer, PG Instruments , 2,000 rpm for 5 minutes. Physiological markers of the liver United Kingdom) at 570 and 595 nm. +e antiproliferative [blood urea nitrogen (BUN), glutamate oxaloacetate effect was expressed as the percentage of the control transaminase (GOT), glutamate pyruvate transaminase according to Ferreira et al. [12]. (GPT), alkaline phosphatase (ALP)] and kidneys (creati- nine) were evaluated according to Labmax Plenno Labtest . Subsequently, all animals were euthanized to dissect out the 2.3.2. Trypan Blue Exclusion Assay. Sarcoma 180 cells liver, kidneys, spleen, stomach, heart, and lungs to estimate (0.5 ×10 cells/mL) plated in 24-well plates were exposed to wet relative weights and for macroscopic analysis. Next, DFMC at 5, 10, and 25 μg/mL. Doxorubicin (Dox, 0.3 μg/ organs were fixed with 10% buffered formalin, processed, mL) was used as a positive control. Cell viability was ex- and cut into small pieces to prepare histological sections amined by the exclusion of trypan blue [24]. Briefly, aliquots (4–7 μm). Staining was carried out with hematoxylin and of 10 μL were collected from DFMC-treated S180 cultures eosin (H&E, Vetec , Brazil). Morphological blind analyses after 72 h of exposure, and viability was separated into viable were performed under light microscopy (Olympus , Japan) blue-marked and nonviable blue-coloured cells in a Neu- by an expert pathologist. bauer chamber under light microscopy (Biosystems , USA). 2.4.2. Determination of Chromosomal Damages. +e femurs 2.3.3. Cytokinesis-Block Micronucleus (CBMN) Assay. were removed and carefully cleaned, and proximal epiphyses Sarcoma 180 cells were plated in 24-well plates were sectioned. Bone marrow samples were collected using (0.5 ×10 cells/mL) and treated with DFMC at 5, 25, and 5 mL syringes filled with 0.5 mL of sterile fetal bovine serum 50 μg/mL. After 44 h at 37 C, cytochalasin B (Sigma Aldrich, (Cultilab , Brazil), centrifuged, and homogenized. Drops of USA, 6 μg/mL) was added, and the cells were reincubated for cell suspension were transferred to slides to prepare smears an additional 28 h. At 72 h, the cultures were transferred to (two slides/animal), fixed and stained by the Leishman tubes and centrifuged at 800 rpm for 5 minutes. +en, the method. All analyses were blindly performed under light supernatant was removed, and the body of the cell bottom microscopy (Olympus , Japan) with magnifications of 200x was enlarged and centrifuged again before the addition of and 400x. We considered micronuclei to be rounded 2 mL of fixing solution (methanol and acetic acid, ratio 5 :1) structures, with a diameter of 1/5 to 1/20 found in young and 3 drops of formaldehyde 37% (Vetec , Brazil). +is erythrocytes and identified by bluish staining. A total of procedure was repeated 3x using fixing solution 3 :1 without 1,000 polychromatic erythrocytes (PCEs) was quantified per formaldehyde. Supernatants were discarded, and 2-3 drops slide (two slides/animal) [28–30]. of cell suspension were dripped onto slides and stained with Giemsa for 5 min [25]. Considering blind examination, a 2.5. Statistical Analysis. Half maximal inhibitory concen- total of 2000 cells by concentration were counted by optical tration (IC ) and their 95% confidence intervals were microscopy at 1000x (1000 cells/slide) to count buds, calculated by nonlinear regression (GraphPad Prisma 9.0, bridges, and micronuclei. Intuitive Software for Science, USA). Statistical differences were evaluated comparing data [mean± standard error of mean (S.E.M.)] by one-way analysis of variance (ANOVA) 2.4. In vivo Assays and Newman–Keuls test as post hoc test (p< 0.05). All in 2.4.1. Assessment of Antitumor Capacity, Physiological Pa- vitro studies were carried out in duplicate (n � 3/concen- rameters, and Histological Aspects. Ten-day-old S180 ascites tration) and represent independent biological evaluations. tumor cells were removed from the peritoneal cavity, counted (6 ×10 cells/mL) and subcutaneously implanted 3. Results into the right hind axillary of healthy Swiss animals. On the next day, they were randomly divided into four groups 3.1. In vitro Antiproliferative Action on Sarcoma 180 Cells: (n � 10 each). DFMC dissolved in dimethylsulfoxide (DMSO Cytotoxicity, Chromosomal Changes, and Cell Death Pattern. 5%, Vetec , Brazil) was intraperitoneally injected at 50 or DFMC and betulinic acid revealed similar in vitro anti- 100 mg/kg/day for 7 days. Negative and positive controls proliferative activity against S180 cells after 72 h of incu- received DMSO 5% and 5-fluoruracil (5-FU, 25 mg/kg/day, bation, with IC values of 29.0 (24.9–33.6) μg/mL and 33.7 Sigma Aldrich , USA), respectively [26]. (30.1–37.6) μg/mL, respectively (p> 0.05, Table 1). After- All animals were anaesthetized on day 8 with ketamine wards, this action was confirmed by trypan blue assay (90 mg/kg)-xylazine (4.5 mg/kg) for cardiac puncture blood (Figure 1), a direct method to detect cytotoxicity, which collection [27] using sterile tubes and heparinize pipettes to showed that all concentrations of DFMC (5, 25, and determine hematological parameters (erythrocytes, leuko- 50 μg/mL) reduced the number of viable cells (48.2± 7.1, cytes, platelets, hemoglobin, and hematocrit) in peripheral 87.6± 1.4, and 98.7± 0.5%, respectively) when compared to blood samples using an automatic analyzer of hematologic the negative control (p< 0.05). cells (SDH-3 Vet Labtest , Brazil). +e absolute count of Morphological analysis of DFMC-treated Sarcoma 180 white cellular subtypes was calculated as the product of its cells did not show significant micronucleus induction 4 Journal of Oncology Table 1: Cytotoxic activity of the dichloromethane fraction and betulinic acid from Mimosa caesalpiniifolia (DFMC) stem bark on primary culture of sarcoma 180 cells after 72 h of exposure evaluated by alamar blue assay. IC (μg/mL) Sample Sarcoma 180 cells R DFMC 29.0 (24.9–33.6) 0.9278 Betulinic acid 33.7 (30.1–37.6) 0.9292 Doxorubicin 1.9 (1.4–2.4) 0.9801 Data are presented as IC values and 95% confidence intervals. Doxorubicin was used as positive control. Experiments were performed in duplicate. 48.2 ± 7.1% 64.1 ± 4.1% 87.6 ± 1.4% * 98.7 ± 0.5% µg/mL C Dox 5 25 50 DFMC Figure 1: +e cell number of viable cells was determined by trypan blue staining and analyzed by light microscopy after 72 h of exposure to the dichloromethane fraction from Mimosa caesalpiniifolia (DFMC) stem bark. +e percentage of viability reduction in relation to the negative control is described above. +e negative control (C) was treated with the vehicle used to dilute the tested substance. Doxorubicin (Dox, 0.3 μg/mL) was used as a positive control. +e results are expressed as mean ± standard error of measurement (S.E.M.) from two independent experiments. p< 0.05 compared to the control by ANOVA followed by student Newman–Keuls test. (4.5± 0.7, 5.5± 2.1, and 4.5± 2.1 for 5, 25, and 50 μg/mL, were observed, which indicated a reduction in proliferation (Figures 3(e)–3(l)). 5-FU-treated tumors showed larger respectively) in relation to the negative control (3.5± 0.7, p> 0.05, Figure 2(a)). On the other hand, bridges (14.6± 3.9 blood vessels and well vascularized sarcomas, similar to and 27.0± (2) and buds (13.8± 3.3) were observed at 25 and those noted in negative control tumors (Figure 3(e)). On the 50 μg/mL and 50 μg/mL (p< 0.05) when compared to the other hand, DFMC-treated Sarcoma 180 tumors treated with negative control (2.0± 1.4 and 5.5± 3.5), respectively. Such 50 and 100 mg/kg/day exhibited poorer peri- and intratumor chromosomal damage was corroborated by morphological quantities of vessels. In such tumors, vascularization was features of apoptosis (213.0± 73.5 and 337.0± 57.9) and partially restricted to the adipose tissue surrounding the necrosis (162.5± 60.1 and 189.5± 40.3) at 25 and 50 μg/mL tumor (Figures 3(i)–3(j)). (p< 0.05, Figure 2(b)) in the presence of cell rarefaction and vacuolization. As expected, Dox increased buds (15.5± 3.5) 3.3. Physiological Parameters. In the next step, we assessed and micronuclei (18.5± 4.9) and caused typical findings of macroscopic and microscopic parameters of key organs and apoptosis (466.0± 101.8) and necrosis (177.5± 3.5) (p< 0.05). the hematological profile of Sarcoma 180-bearing mice after treatment with DFMC. First, we found a reduction in body 3.2. In vivo Antitumoral Activity. Experimentally trans- weight gain in DFMC-treated animals (20.6± 0.8 and planted mice with Sarcoma 180 cells and treated with DFMC 21.4± 1.6 g, for 50 and 1000 mg/kg/day) in a similar way to (50 and 100 mg/kg/day) for 7 days revealed a significant the 5-FU group (20.1± 0.9 g) when compared to the negative control (26.3± 2.2 g, p< 0.05, Table 1). Wet relative weight reduction in tumor growth [(0.28± 0.04 g (64.8± 5.3%) and 0.16± 0.07 g (80.0± 8.4%)] when compared to the negative reduction of spleens was noted in both doses of DFMC (0.2± 0.08 and 0.2± 0.03 g/100 g of body weight) and in 5- control (0.80± 0.13 g, respectively). Tumor reduction was also noted in the positive control group treated with 5-FU FU-treated animals (0.2± 0.04 g), but liver decrease was [0.11± 0.03 g (82.8± 4.2%)] (p< 0.05, Table 2). observed in 5-FU-treated animals only (4.7± 0.1 g) in +e negative control group showed characteristics of comparison with the negative group (0.4± 0.04 g and malignant neoplasms consisting of round and polyhedral 6.0± 0.4 g, respectively, p< 0.05). cells, anisocariosis, binucleation, mitoses, and different Hematological analysis of DFMC-treated animals showed degrees of cell and nuclear pleomorphism, chromatin neutrophilia (33.8± 3.2%), lymphocytopenia (61.5± 3.6%), a condensation, and extensive areas of muscle invasion reduction in eosinophils (0.4± 0.2%), and a slight increase in (Figures 3(a)–3(d)). Tumor samples from 5-FU 25 mg/kg/ GOT levels (315.3± 8.9 U/mL) (p< 0.05, Table 3). Animals exposed to 5-FU showed intense leukopenia (1.6± 0.3/mm day and FDCM 50 and 100 mg/kg/day also revealed the ) typical morphology of neoplastic cells, although rare mitoses due to declines in neutrophils (12.9± 1.3%), monocytes Cell number (×10 /mL) Journal of Oncology 5 20 * C Dox 5 25 50 µg/mL DFMC Buds Micronucleus Bridges (a) 200 * C Dox 5 25 50 µg/mL DFMC Apoptosis Necrosis (b) Figure 2: Ex vivo chromosomal changes and cell death pattern in sarcoma 180 cells determined by micronucleus assay with cytokinesis block after 72 h exposure to the dichloromethane fraction from Mimosa caesalpiniifolia (DFMC) stem bark. +e negative control (C) was treated with the vehicle used to dilute the tested substance (DMSO 0.1%). Doxorubicin (Dox, 0.3 μg/mL) was used as a positive control. +e results are expressed as mean± standard error of measurement (S.E.M.) from two independent experiments. p< 0.05 compared to the control by ANOVA followed by student Newman–Keuls test. Table 2: Effect of the dichloromethane fraction from Mimosa caesalpiniifolia (DFMC) stem bark on the relative weight of key organs and on the tumor growth of sarcoma 180-bearing swiss mice after 7 days of intraperitoneal treatment. Dose (mg/kg/ Mice weight Liver Kidney Spleen Stomach Lungs Tumor inhibition Substance Tumor (g) day) (g) g/100 g body weight (%) Negative — 26.3± 2.2 6.0± 0.4 1.1± 0.1 0.4± 0.04 1.0± 0.1 0.8± 0.1 0.80± 0.13 — control ∗ ∗ ∗ ∗ ∗ 5-FU 25 20.1± 0.9 4.7± 0.1 1.2± 0.1 0.2± 0.04 1.1± 0.1 0.8± 0.1 0.11± 0.03 82.8± 4.2 ∗ ∗ ∗ ∗ 50 20.6± 0.8 5.8± 0.2 1.2± 0.1 0.2± 0.08 1.1± 0.1 1.0± 0.2 0.28± 0.04 64.8± 5.3 DFMC ∗ ∗ ∗ ∗ 100 21.4± 1.0 5.9± 0.2 1.3± 0.1 0.2± 0.03 1.2± 0.5 0.8± 0.1 0.16± 0.07 80.0± 8.4 Values are means± S.E.M. (n � 10 animals/group). +e negative control was treated with the vehicle used to dilute the drug (DMSO 5%). 5-Fluorouracil (5-FU) was used as positive control. p< 0.05 compared with the negative control by ANOVA followed by Newman–Keuls test. (0.6± 0.2%) and eosinophils (0.6± 0.3%) compared to the from these groups. Livers did not exhibit hyperplasia, he- animals from the negative group (5.1± 0.4/mm , 18.8± 2.8%, mosiderin pigments, infiltration of leukocytes, cell swelling, 1.8± 0.3% and 1.8± 0.4%, respectively, p< 0.05). portal congestion, or areas of necrosis, although micro- esteatosis was detected in all groups (Figure 4(a)). Kidneys present no swelling, tubular degeneration, vascular congestion, 3.4. Histological Alterations. Animals from the negative or necrosis focus (Figure 4(b)); in hearts, there were no areas of control group and treated with DFMC (50 and 100 mg/kg/day) degeneration or fibroblasts proliferation and striations were did not show signs of toxicity, with similarity among organs clearly visible (Figure 4(c)); lungs showed bronchioles and Events (2000 cells) Events (2000 cells) 6 Journal of Oncology AB C D EF G IJ K L Muscular invasion Chromation condensation Intratumoral vessels Binucleation Mitosis Microenvironment vessels Figure 3: Morphology of sarcoma 180 tumor cells from swiss mice after 7 days of treatment with dichloromethane fraction from Mimosa caesalpiniifolia stem bark. Animals were treated by intraperitoneal injection (50 mg/kg/day: g, h, and i; 100 mg/kg/day: j, k, and l). +e negative control was treated with the vehicle used to dilute the substance (DMSO 5%: a–d). 5-Fluorouracil was used as a positive control (e and f). Hematoxylin-eosin staining. Light microscopy magnification, 100x-400x. Table 3: Hematological and biochemical parameters of mice intraperitoneally treated with dichloromethane fraction from Mimosa caesalpiniifolia stem bark for 7 days. Dichloromethane fraction from Mimosa caesalpiniifolia Parameters Negative control 5-FU 25 mg/kg/day 50 mg/kg/day 100 mg/kg/day Erythrocytes (mm ) 4.5± 0.2 4.4± 0.2 5.0± 0.1 4.9± 0.2 Hemoglobin (g/dL) 13.6± 0.7 13.3± 0.8 15.4± 0.4 15.0± 0.7 Hematocrit (%) 40.7± 2.3 40.1± 2.3 46.3± 1.1 44.9± 2.2 VCM (fL) 90.8± 0.5 90.6± 0.6 91.8± 0.3 91.6± 0.5 HCM (pg) 30.2± 0.2 30.2± 0.2 30.5± 0.1 30.5± 0.2 CHCM (g/dL) 33.3± 0.1 33.1± 0.1 33.2± 0.1 33.3± 0.1 Platelets (mm ) 3.6± 0.5 2.9± 0.2 3.4± 0.2 3.4± 0.2 3 ∗ Total leukocytes (mm ) 5.1± 0.4 1.6± 0.3 5.4± 0.6 4.9± 0.7 ∗ ∗ Neutrophils (%) 18.8± 2.8 12.9± 1.3 23.3± 4.1 33.8± 3.2 Rods (%) 0.4± 0.2 0.4± 0.3 0.6± 0.2 1.8± 0.7 Lymphocytes (%) 77.3± 3.0 85.6± 1.8 73.7± 4.1 61.5± 3.6 Monocytes (%) 1.8± 0.3 0.6± 0.2 1.7± 0.6 2.6± 0.8 ∗ ∗ ∗ Eosinophils (%) 1.8± 0.4 0.6± 0.3 0.7± 0.3 0.4± 0.2 Basophils (%) 0.0 0.0 0.0 0.0 GOT (U/mL) 286.9± 5.8 303.2± 7.6 280.8± 9.1 315.3± 8.9 GTP (U/mL) 158.8± 2.6 157.5± 4.3 161.6± 5.0 156.3± 1.1 ALP (U/L) 112.3± 5.5 131.2± 9.8 101.2± 3.4 93.8± 6.6 Creatinine (mg/dL) 0.5± 0.05 0.5± 0.08 0.4± 0.01 0.4± 0.04 BUN (mg/dL) 48.9± 4.2 37.7± 2.4 41.3± 6.6 42.8± 3.1 MCH, mean corpuscular hemoglobin; MCV, mean corpuscular volume; MCHC, mean corpuscular hemoglobin concentration; BUN, blood urea nitrogen; GOT, glutamate oxaloacetate transaminase; GPT, glutamate pyruvate transaminase; ALP, alkaline phosphatase. Values are means± S.E.M. (n � 10 ani- mals/group). +e negative control was treated with the vehicle used to dilute the drug (DMSO 5%). 5-Fluorouracil (5-FU) was used as positive control. P< 0.05 compared with the negative control by ANOVA followed by Newman–Keuls test. visible alveolar spaces, absence of mono and polymorpho- hemorrhagic streaks, a cardiac region with a keratinized nuclear cells or areas of necrosis (Figure 4(d)); stomachs squamous lining, no changes in chorion and easy visualization showed normal mucosa and submucosa, absence of of parietal and main cells (Figure 4(e)). Spleens showed Journal of Oncology 7 Kuper cell ff Tubular space Centrilobular vein Capsular space Glomerulus Arterioles Liver Duct Microesteatosis Proximal tubule (a) (b) Normal fibrocytes Pneumocyte type II Transverse streaks Pneumocyte type I Intercalated discs Capillary Nucleus Alveolar sac (c) (d) Muscus cells Gastric glands Gastric pits (e) Figure 4: General morphology of livers (a), kidneys (b), hearts (c), lungs (d), and stomachs (e) from Swiss mice after 7 days of treatment with dichloromethane fraction from Mimosa caesalpiniifolia stem bark (50 or 100 mg/kg/day) or vehicle used to dilute the substance (DMSO 5%). Important changes among these groups were not observed. Hematoxylin-eosin staining. Light microscopy magnification, 400x. megakaryocytes and hemosiderin pigments in all groups. indisputable advantage when compared to traditional Disorganization of lymphoid follicles and relative reduction of treatments based on surgery and monochemotherapy, the white pulp were observed in the 5-FU (Figure 5(b)) and making it possible to cure neoplasms such as acute child- DFMC-treated animals (Figures 5(c) and 5(d)). On the other hood leukemia, Hodgkin and non-Hodgkin’s lymphomas, hand, 5-FU-treated animals showed slight hepatocyte swelling and germ cell tumors [31, 32]. However, the great hetero- and suggestion of mild changes in fatty metabolism since geneity of tumor cells makes treatment difficult and facili- macroesteatosis was noted, and kidneys presented swelling of tates the manifestation of resistance [33], which stimulates tubular cells and foci of atrophic glomeruli (results not shown). the search for new chemotherapeutic agents. Initially, the antiproliferative action of DFMC was eval- uated in primary cultures of Sarcoma 180 cells. In vitro cy- 3.5. In vivo Chromosomal Damage. DFMC increased totoxicity tests in cell cultures are important for the evaluation micronucleated polychromatic erythrocytes in the bone of antitumor agents, and at least during the screening phase, marrow of mice in a dose-dependent manner (50 and they have reduced in vivo tests on animals. In addition, they 100 mg/kg/day: 11.5± 0.2 and 26.0± 2.1, respectively) are widely used as alternative methods to pharmacological compared to the vehicle group (2.8± 0.2, p< 0.05). As ex- tests on isolated organs [26, 34]. Herein, DFMC and its pected, 25 mg/kg/day 5-FU caused clastogenic effects majority compound betulinic acid revealed similar cytotoxic (14.0± 0.1, p< 0.05). capacity on S180 cells by Alamar blue assay, whose action was confirmed by cell viability reduction in trypan blue exclusion tests. Some reports, including the American National Cancer 4. Discussion Institute (NCI-USA) [35], suggest that IC values around In the last century, the development of cytotoxic agents has 30 μg/mL are a suitable outcome to consider extracts and revolutionized anticancer therapy. Adjuvant treatments with fractions promising substances for further purification and antiproliferative substances have demonstrated an biological studies [12, 15]. Recently, we reported that DFMC 8 Journal of Oncology White pulp White pulp Hemosiderin pigments Megakaryocytes Megakaryocytes Hemosiderin pigments Trabeculae Red pulp (a) (b) Central arteriole Disorganization of follicles Megakaryocytes Megakaryocyte Hemosiderin pigments Disorganization of follicles (c) (d) Figure 5: Spleen morphology of Swiss mice after 7 days of treatment with dichloromethane fraction from Mimosa caesalpiniifolia stem bark (50 mg/kg/day (c); 100 mg/kg/day (d)), vehicle used to dilute the substance DMSO 5% (a) or 5-fluorouracil 25 mg/kg/day (b). Hematoxylin- eosin staining. Light microscopy magnification, 400x. has higher cytotoxic action against different types of tumor block micronucleus (CBMN) assays were performed to tissues (promyelocytic leukemia, HL-60; glioblastoma, SF- measure micronuclei quantification and DNA damage in 295; ovarian, OVCAR-8; colon, HCT-116) than hexane and mammalian cell cultures [28]. Apart from the evaluation of water extracts. DFMC did not produce hemolysis and showed micronuclei, the CBMN cytome assay allows the assessment higher potential as a cytotoxic agent than betulinic acid for the of other relevant biodosimetric markers: nucleoplasmic SF-295 and HL-60 lines [20, 36], corroborating the findings bridges, nuclear buds, proportion of dividing cells (pa- rameter of cytostasis), and cells undergoing apoptosis and described here for S180 cells. Phytochemical investigation of extracts from Mimosa necrosis (parameters of cytotoxicity). +erefore, this tech- nique was updated to detect chromosomal breaks, DNA species revealed the existence of terpenes, flavonoids, ste- roids, phenols (especially tannins), and fatty acid derivatives rearrangements, chromosomal losses, cytostasis, and to in different parts of the plant (leaves, fruits, flowers, separate types of cell death [25, 28, 51, 52]. +erefore, for the branches, and stem bark) [36–40], mainly betulinic acid, first time, an increase in chromosomal damage represented lupeol, phytol, lactic acid, α-tocopherol, stigmasterol, β-si- by (i) nucleoplasm bridges: a biomarker of dicentric chro- tosterol, sitostenone, and stigmasta-4,22-dien-3-one, which mosomes, resulting from the fusion of the final telomeres had been identified in dichloromethane, ethanolic, and after DNA double-strand breaks or DNA misrepair/rear- hexane fractions of leaves and barks from M. caesalpiniifolia rangements; (ii) buds: a biomarker of gene amplification and [15, 36, 40], suggesting that the antiproliferative potential of originating from interstitial or terminal acentric fragments; and (iii) morphological features of apoptosis and necrosis in DFMC may be attributed, at least in part, to its chemo- preventive action. In this context, Silva et al. [15] stated the S180 cells at higher concentrations of DFMC was noted. Meanwhile, both doses of DFMC also induced the emer- scavenger activity of M. caesalpiniifolia extracts, whose presence of phenolic compounds was confirmed by ultra- gence of micronucleated polychromatic erythrocytes in bone violet-visible spectroscopy and thin-layer chromatography. marrow. Previously, Silva et al. [23] reported an ethanolic Betulinic acid, a naturally occurring pentacyclic tri- extract from M. caesalpiniifolia leaves with maximum cy- terpenoid, is the main compound in the fraction (∼70.3%) totoxicity on breast carcinoma MCF-7 cells at 320 μg/mL [15, 23], and both samples (DFMC and isolated molecule) and morphological changes suggestive of apoptosis, in- have similar bioactivity on S180 cells (p> 0.05), confirming cluding DNA fragmentation and nuclear chromatin reports about the antiproliferative action of betulinic acid in condensation. many types of cancers [41–50]. Recently, we also showed that micronuclei formation and changes indicating mutagenic index in DFMC-treated To complement the ex vivo cytotoxic analysis on S180 tumor cells and in vivo pharmacological safety, cytokinesis- roots were not detected, although this fraction has inhibited Journal of Oncology 9 also essential data about the pharmacokinetics profile, growth of Allium cepa roots and increase amount of bridges in dividing meristematic cells, which indicates capacity for therapeutic window, and pharmacological safety, including systemic and genetic toxicology [58, 59]. +ese assessments mitotic index reduction as seen as dropping of cells at metaphase, anaphase, and telophase phases and cycle arrest allow the exclusion of undesirable drug candidates and save at prophase [15]. Regardless, it is likely that DNA/chro- time, material and human resources. In the case of plant mosomal damage is a sign of nonselective mechanism(s) in toxicity/poisoning, its harmful action must be proven ex- tumor or normal dividing cells. +erefore, in vitro (bridges perimentally. For humans, this experimental reproduction and buds) and in vivo (micronucleus) clastogenic findings should be carried out in the same animal species, naturally led to cell cycle arrest as a “cellular escape” from death, affected, or related species (e.g., mice and rats), although different susceptibilities to the effects of toxic herbals among mainly if we consider the antiproliferative action of DFMC on human normal leukocytes well [15]. species are a common mark [60, 61]. Acute signs of systemic toxicity are loss of body mass and Indeed, antineoplastic agents induce DNA strand breaks in mammalian cells, as seen with inhibitors of topoisomerase expansion or involution of key organs in mammals exposed to an investigational drug [62]. Weight loss is one of the I (camptothecin) and topoisomerase II (etoposide) [53] and 5-FU. 5-FU is a widely used antimetabolite to treat breast most common side effects after chemotherapy cycles with 5- adenocarcinomas and cancers of the gastrointestinal tract FU or doxorubicin, since the gastrointestinal system is one and head and neck due to its inhibitory action on the enzyme of the main nonspecific targets of nontarget antiproliferative thymidylate synthase [54], among other mechanisms, de- agents, causing seasickness, suppression of appetite, vom- spite its unblemished in vivo clastogenic activity [55]. iting, and diarrhea [63]. Loss of body weight and reduction However, genotoxicity does not mean mutagenicity because of spleens were macroscopic manifestations found in the 5- FU- and DFMC-treated groups, but signs of diarrhea were some genome injuries are biochemically fixed, which indi- cates that antineoplastic acute toxic consequences (e.g., not seen in the DFMC-treated groups. Spleen diminution is another very common side effect found in S180-bearing inhibition of growth and cell division) are not automatically linked to chromosomal loss/impairments [56]. mice under experimental treatment with promising anti- tumoral substances [26, 64] and reflects lymphocytopenia +e cytotoxic activity on cancer cells using in vitro models may not reflect in vivo findings, since the latter seen in 5-FU- and DFMC-treated groups and strong leu- considers pharmacokinetic and pharmacodynamic vari- kopenia in 5-FU-treated mice, which was confirmed by ables, such as ligand binding to specific receptors, down- disorganization of lymphoid follicles and size reduction of stream cascade, involvement of second messengers, water/ white pulps. lipid solubility, bioavailability, first-pass metabolism, and In vivo toxicological studies with DFMC were not found in renal excretion [57, 58]. +erefore, combining these two the literature, but oral subacute treatment of rats for 32 days with 750 mg/kg/day ethanolic extract from M. caesalpiniifolia types of scientific tools is appropriate for a more complete assessment of a substance with antiproliferative action. For leaves caused weight loss, hepatomegaly, and an increase in adrenal and pituitary glands [40], but serum biochemical the first time, the amazing antitumor action of a dichloromethane fraction from M. caesalpiniifolia stem bark evaluation (alkaline phosphatase, GOT, urea, and creatinine) on in vivo proliferating Sarcoma 180 cells was demonstrated. did not identify renal or liver changes. On the other hand, we In vivo studies have already shown that betulinic acid in- noted that the 100 mg/kg/day DFMC-treated group revealed a hibits the growth of human ovarian IGROV-1 carcinoma slight but significant increase in GOT. xenographic tumors at 100 mg/kg/day and increases the Transaminases (GOTand GTP) are found in all human survival rate of mice [46]. systems and many organs, but they are more present in the No specific changes were noted during histopathological cytoplasm (100% GTP/20% GOT) or mitochondria (80% analysis of the Sarcoma 180 tumors [34], but it is important GOT) of hepatocytes, since they catalyze transamination reactions, working central providers of secondary me- to emphasize that local vascularization from DFMC-treated animals was predominantly confined to the adipose tissue tabolites to the citric acid cycle. Any type of liver injury may sensibly increase serum GTP concentrations, a classic surrounding the tumors. +ese unexpected findings were not described before and suggest that the fraction interferes biomarker to assess acute or chronic hepatic damage, but with the cell cycle of Sarcoma 180 cells and inhibits an- its origin can have kidney, heart, or muscle reasons be- giogenesis, which obviously alters the stromal environment, cause these organs also possess higher GTP concentra- such as the local pH, partial pressure of oxygen and carbon tions in comparison with other tissues [65]. On the other dioxide, input of nutrients/growth factors, and cleaning of hand, GOT is more abundant in heart, skeletal muscle, metabolic residues [57], all essential primary conditions for kidneys, brain, and red blood cells [66], with lower concentrations in skeletal muscle and kidney. Although cellular division and tumor growth. Molecular studies are underway to confirm such antiangiogenic potential. +ese GTP is more specific for detecting liver damage, ischemic or toxic damage to zone 3 of the hepatic acinus may data corroborate our findings about the biomedical potential of M. caesalpiniifolia and inspired us to assess the phar- change GOT levels since this region has greater GOT concentrations [65]. macological safety profile of the fraction, taking into con- sideration its promising phytotherapy properties. Histological changes were not found in livers from +e development of new (phyto)pharmaceutical prod- DFMC-treated animals. +us, it is probable that higher ucts includes not only pharmacodynamic discoveries but levels of GOT may be associated with muscle damage and/ 10 Journal of Oncology In vitro antiproliferative and clastogenic effects upon S180 cells Betulinic acid-rich Slight physiological changes: fraction from Mimosa loss of weight, reduction of spleen, CO H lymphocytopenia and neutrophilia, caesalpiniifolia stem bark GOT increasing, MN in bone marrow HO In vivo tumor reduction ranging from 64.8 to 80 % Figure 6: Summary of the antiproliferative, genotoxic, antitumoral, and toxicological effects of a betulinic acid-rich fraction from Mimosa caesalpiniifolia stem bark. or trauma after continual intraperitoneal injections be- Conflicts of Interest cause this procedure can result in aminotransferase re- +e authors declare that there are no conflicts of interest lease, and an increase in GOT is common in such regarding the publication of this paper. situations [66]. +e majority of clinically available anticancer medica- tions provoke strong side effects, especially suppression of Acknowledgments bone marrow and immune response, toxicity on hepato- cytes, cardiac myocytes and enterocytes, mucositis, weight Paulo Michel Pinheiro Ferreira is grateful to the public and hair loss (incidence of 65%), opportunistic infections, Brazilian agency “Conselho Nacional de Desenvolvimento seasickness, vomiting, chemotherapy-related anorexia, pe- Cient´ıfico e Tecnologico” ´ [CNPq (#303247/2019-3)] for his ripheral neuropatia, and tiredness [33, 63, 67–69], whose personal scholarship. +e authors also thank Leane Brunelle types and intensity depend on the mechanism(s) of action dos Santos Alves for technical assistance and the Post- and idiosyncratic reactions. 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