Genotoxicity Assessment and Protective Effect of Anogeissus leiocarpus Roots against Cyclophosphamide-Induced DNA Damage In Vivo

Background Belonging to the family of Combretaceae, the roots of Anogeissus leiocarpus are traditionally used to treat diabetes, wounds, infections, pain, and gastrointestinal diseases. To our knowledge, no genotoxicity assessment of the plant was reported. Hence, this study was designed to evaluate the potential genotoxic and protective effects of extract of Anogeissus leiocarpus roots using the micronucleus test on mice bone marrow cells in vivo. Methods Three different concentrations (250, 500, and 1000 mg·kg−1) of hydroalcoholic extract of roots of A. leiocarpus were administered daily for 7 days per os to mice, and the genotoxicity was induced by the administration ip of cyclophosphamide. Genotoxicity and cytotoxicity were evaluated by counting, respectively, the number of micronucleated polychromatic erythrocytes and polychromatic erythrocytes to total erythrocytes in the bone marrow of mice. Results The administration of A. leiocarpus did neither increase the ratio of the polychromatic erythrocyte (PCE) nor the frequency of micronucleated PCE (MNPCE) significantly in the bone marrow cells of the mice, compared to the vehicle control animals. However, a significant increase in the incidence of MNPCE in the bone marrow cell of the cyclophosphamide-treated mice was found. Moreover, in the groups treated with the total extract of A. leiocarpus at different doses plus cyclophosphamide, there was a significant decrease (p < 0.0001) in MNPCEs compared to the positive controls, in a dose-dependent manner. Conclusion This first finding reports that the extract of A. leiocarpus was neither genotoxic nor cytotoxic. However, it shows a protective effect against the genotoxicity and cytotoxicity induced by cyclophosphamide.


Introduction
Cyclophosphamide is an alkylating chemotherapeutic drug extensively used to treat many types of cancers including lymphoma, leukemia, breast, ovarian, and lung carcinomas [1]. However, its clinical use can lead to various organ toxicities [2,3]. Cyclophosphamide has been reported to induce dominant lethal mutation, micronuclei, DNA damage, and generation of free radicals or reactive oxygen species (ROS), chromosomal aberrations, sister chromatid exchanges, and gene mutations. is can lead to a multitude of pathological conditions as mutagenicity, carcinogenicity, teratogenicity, myelosuppression, immunosuppression, reproductive toxicity, cardiac toxicity, lung toxicity, and urotoxicity [4][5][6][7][8].
e clinical efficacy of cyclophosphamide is restricted due to its undesired toxicities in normal cells. erefore, it is important to prevent normal cell DNA damage induced by cyclophosphamide [9]. Plants may offer new alternatives to these limited therapeutic options. erefore, it has been an increased scientific interest in the study of materials from a plant source as an anticancer compound [10,11]. Many studies in the literature have reported the expressive anticarcinogenic and antimutagenic activities of plants that contained phenolic compounds, flavonoids, and tannins due to their antioxidant properties [12][13][14].
Anogeissus leiocarpus, a plant of the Combretaceae family, is widely used in traditional medicine in Togo to treat various pathologies wounds, infections, pain, gastrointestinal diseases, and diabetes [15,16]. e phytochemical constituents of Anogeissus leiocarpus were found to be polyphenolic compounds, flavonoids, and triterpenes [17,18].
Our previous works had reported the antihyperglycemic, lipid-lowering, and antioxidant activities of the total extract and fractions of roots of Anogeissus leiocarpus [19,20]. Administered at daily repeated doses of 500 mg·kg −1 and 1000 mg·kg −1 , the hydroalcoholic extract of A. leiocarpus is found to be nontoxic in rats following either a single dose or daily repeated doses during 28 days in vivo [21].
Despite the large use of roots of Anogeissus leiocarpus, no report on its genotoxicity assessment is available in the literature. us, based on the pharmacological activity of this plant, especially its strong antioxidant properties, this current study was investigated to evaluate the potential genotoxic as well as the protective effects of hydroalcoholic extract of Anogeissus leiocarpus roots using the micronucleus test on mice bone marrow cells in vivo.

Animals.
Male ICR mice (30 ± 5 g) were kept in standard environmental conditions (temperature 24-25°C, relative humidity, and a 12 t/12 h light-dark cycle) and fed with standard rat diet and water ad libitum. Female mice were excluded from the study because of their cyclic hormonal variations.
is study has been approved by the Ethics Committee of the University of Lomé, a branch of the National Ethics Committee for control and supervision of experiments on animals (NSBM/UL/14/NS0004).

Plant Material.
Roots of Anogeissus leiocarpus were harvested in Tsévié, Zio (Togo). A voucher specimen was identified and deposited in the herbarium of the Laboratory of Botany and Plant Ecology under the number TOGO 15483. Roots of Anogeissus leiocarpus were cleaned out with water, cut into small pieces, dried at the Animal Physiology laboratory at 22°C, and then reduced into powder using the Mill omas Scientific TM .

Plant Extraction.
About 400 g of roots of Anogeissus leiocarpus were extracted in water/ethanol (5 : 5) for 72 hours. e crude extract was filtered on Whatman paper and evaporated in a vacuum at 45°C using a rotary evaporator (IKA® RV 10 digital). e yield of the dry extract was 5.68% and was stored at 4°C [18].

Genotoxicity and Antigenotoxicity Assessment by the Micronucleus Test of Roots of Anogeissus leiocarpus in Mice
Bone Marrow. OECD 474 test guidelines [22] with slight modifications for the mammalian erythrocyte micronucleus test states was used for this test on mice, divided into 8 groups of 5 animals. ree different concentrations of A. leiocarpus have been tested for potential genotoxic effects and cytotoxic activities in vivo in bone marrow cells of mice. e choice of doses of 250, 500, and 1000 mg·kg −1 was based on the therapeutic dose of A. leiocarpus that has been determined in our previous studies [19,20].

Experimental Design.
To evaluate the possible toxic effect of the plant, the animals were divided into 5 groups as follows: group 1 received distilled water and served as a negative control. Group 2, a positive control received a dose of 100 mg·kg −1 of cyclophosphamide I.P. ree doses of 250 mg kg −1 bw, 500 mg kg −1 bw, and 1000 mg·kg −1 bw of hydroalcoholic extract of Anogeissus leiocarpus dissolved in distilled water had been administered daily for 7 days per os, respectively, to mice of groups 3, 4, and 5.
In order to detect the protective effect of Anogeissus leiocarpus, three doses of 250 mg·kg −1 bw, 500 mg·kg −1 bw, and 1000 mg·kg −1 bw of Anogeissus leiocarpus had been administered daily during 7 0 days per os, respectively, to mice of groups 6, 7, and 8. After 7 days of daily pretreatment, mice of groups 6, 7, and 8 received cyclophosphamide intraperitoneal (i.p.) at a dose of 100 mg·kg −1 bw. irty hours after injection, the animals were anesthetized using light diethyl ether and sacrificed by cervical dislocation. All mice were observed daily for signs of toxicity during the treatment period. e bodyweight of each mouse was measured twice: before administration of the extracts and before sacrifice.

Preparation of Slides.
According to the method used by [23], with slight modifications, the femur was immediately removed. e epiphyses were cut, and the bone marrow was flushed out using 1 mL saline solution (NaCl 0.9%). e supernatant was discarded after 7 minutes of centrifugation. Formaldehyde (4%) in distilled water was added to preserve the cytoplasm. e cell suspension was softly smeared on the surface of the slide. Dried slides were fixed in May Grunwald for 2 min and stained with 5% Giemsa solution for 30 minutes. Stained slides were analyzed under the light microscope at the 1000 × magnification. Slides were observed using a binocular microscope (zigzag orientation) type "Olympus" and marking Є with a 100 × immersion objective. Images were taken directly on the laptop connected to the ocular camera type 049002-VGA (Germany) Є integrated into the microscope.

Micronucleus and Erythrocyte Counting.
A total of 5000 polychromatic erythrocytes (PCEs) per animal were scored for the incidence of micronucleated immature erythrocytes.
e frequency of micronucleated polychromatic erythrocytes (MNPCEs) was expressed as a percentage. Besides, the number of PCE was counted in 1000 total number of erythrocytes (TE � PCE + NCE), and a ratio between PCE and TE represented the frequency of PCE.
Analysis. Data were expressed as mean ± SEM (standard error of the mean) using the GraphPad Prism 7 software. Statistical differences between groups were determined by ANOVA followed by the Bonferroni test and considered significant for p < 0.05. e administration of A. leiocarpus at the doses of 250, 500, and 1000 mg·kg −1 did increase neither the ratio of the polychromatic erythrocytes to total erythrocytes (PCEs%) in the bone marrow nor the frequency of micronucleated PCE (MNPCE) significantly in the bone marrow cells of the mice, compared to the vehicle control animals (Tables 2 and 3).

Evaluation of Genotoxic
However, a significant (p < 0.0001) increase up to 69% in the incidence of MNPCE in the bone marrow cell of the cyclophosphamide-treated mice was found (Figure 1). e results showed no clastogenic/aneugenic effects of Anogeissus leiocarpus.

Antigenotoxic Effect of Total Extract of Anogeissus leiocarpus.
In the groups of animals which were pretreated with the total extract of A. leiocarpus (250, 500, and 1000 mg·kg −1 ) and then received cyclophosphamide, there was a significant reduction (p < 0.0001) in MNPCEs of 34, 44, and 62%, respectively, in a dose-dependent manner. is reduction of mutagenic effects was observed about the positive control group, which received cyclophosphamide without previous administration of the extract.

Discussion
e in vivo micronucleus test, first developed in mouse bone marrow erythrocytes, is one of the genotoxicity tests recommended by international regulatory agencies and government institutions for the evaluation of new substances [24]. Micronuclei (MN) are extranuclear bodies that contain damaged chromosome fragments and/or whole T.E 250 + C, 500 + C, and 1000 + C, pretreated, respectively, with the total extract at 250, 500, and 1000 mg·kg −1 and received cyclophosphamide at 100 mg·kg −1 . Bodyweight was recorded at the beginning of the experimentation and at the end. Bodyweight gain � ending bodyweight-bodyweight at the beginning.  Journal of Toxicology 5 chromosomes that were not incorporated into the nucleus after cell division. Direct DNA damage or breakage, chromosomal aberrations, mitotic apparatus dysfunctions, and interference with DNA synthesis are the possible explanations of MN formation [25,26]. When a bone marrow erythroblast turns into an immature erythrocyte (polychromatic erythrocyte, PCE), the main nucleus is expelled, and any micronuclei formed may remain in the cytoplasm. Detection of micronuclei in these cells is facilitated by the absence of the main nucleus. In this study, genotoxicity, prevention of genotoxicity, and general cytotoxicity were evaluated in mice treated with the total extract of Anogeissus leiocarpus.
Prior to the micronucleus test, our study reported no abnormalities in the general behavior of the animals in the different treatment groups compared to the negative controls throughout the treatment. No difference was also observed between the bodyweight of the mice (at the beginning, and the end of treatment) and between the bodyweight gain of the treated groups compared to the controls. e mutagenic potential of mutagen is estimated by the number of micronucleated polychromatic erythrocytes (MNPCE) in the bone marrow of rodents. An increase of the incidence of micronucleated polychromatic erythrocytes (MNPCE) in treated animals is indicative of induced chromosomal damage [22].
In our study, the administration of the total extract at different doses had not provoked any significant increase of the frequency of micronucleated PCE (MNPCE) in the bone marrow cells of the mice, compared to the vehicle control animals. is demonstrated a nongenotoxic effect of Anogeissus leiocarpus. As expected, cyclophosphamide (the positive control) induced a 69% increase in MNPCE in their bone marrow cell compared to the vehicle control animals.
Our results are consistent with other studies in the literature [24,27].
Cyclophosphamide is a well-known anticancer agent with alkylating properties that induce gene mutations, chromosomal aberrations, micronuclei, and chromatid exchanges in somatic cells [25,28,29]. As a matter of fact, acrolein is one of the metabolites of cyclophosphamide that induces oxidative stress which leads to DNA damage of normal cells and toxicities to various target organs. Acrolein rapidly enters the cell and activates the intracellular reactive oxygen species and nitric oxide production, leading to peroxynitrite formation which ultimately damages the lipids, proteins, and DNA inside the cell [30].
In order to confirm the presence or absence of cytotoxicity, polychromatic erythrocytes (PCE) were determined in 5000 erythrocytes (PCE + NCE) by the ratio PCE/NCE. When bone marrow cells proliferation is affected by a toxic substance, a decrease in PCE/NCE ratio occurred reflecting bone marrow toxicity and cell depression [22]. e administration of the extract of Anogeissus leiocarpus showed no significant decrease in the PCE/ PCE + NCE ratio in the treated groups compared to the negative control group. is suggests a noncytotoxic activity of the total extract of roots of Anogeissus leiocarpus on mouse bone marrow cells.
Moreover, in the groups of mice treated with the extract plus cyclophosphamide, the extract has shown a protective action against genotoxicity induced by cyclophosphamide in the bone marrow. Compared to positive controls, the number of MNPCEs decreased with the dose of extract administered. In fact, at the dose of 1000 mg·kg −1 , the incidence of MNPCE was 1.39. 10-3 compared to 2.42. 10-3% at the dose of 500 mg·kg −1 .
e major components of Anogeissus leiocarpus are polyphenolic compounds, flavonoids, and triterpenes [17,18]. e antigenotoxic property of the plant may be related to its phytochemical components. In our earlier phytochemical and antioxidant studies, in vitro, the total extract had the ability to scavenge free radicals, reduce metal, and possessed strong total antioxidant activity. is strong antioxidant was due to phenolic compounds in the extract [20].
One of the important classes is flavonoids (quercetin, rutin) which exert their genoprotection by chelating the divalent cations, scavenging free radicals, and modulating the enzymes responsible for bioactivation of genotoxic agents and detoxification of their reactive metabolite [31].
Ellagic acid, a naturally occurring plant polyphenol, was evaluated for its antigenotoxicity and antioxidant efficacy against the cyclophosphamide-induced renal oxidative stress and genotoxicity in Swiss albino mice [32]. us, the presence of this polyphenol in our plant may be involved in preventing free radical-mediated cytotoxicity and lipid peroxidation.
Many studies in the literature exhibited a protective effect against the genotoxicity of triterpenoids and tannins in natural products [33,34].

Conclusion
Hence, to our knowledge, this is the first published study that demonstrates that the hydroalcoholic extract of roots of Anogeissus leiocarpus is not genotoxic and not clastogenic at the concentrations used. In addition, the extract has the ability to prevent chromosomal damages caused by cyclophosphamide. Further studies are needed to investigate the molecular modes of action of the extract/compound (s) by Western blotting, real-time RT-PCR, fluorescence microscopy, and expression analyses for a better understanding and safe use of roots of A. leiocarpus.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
AEM designed, performed the experiments, analyzed data, and wrote the draft manuscript. PL-E approved the design, analyzed data, and revised the draft manuscript. AD read and revised the manuscript. KE-G coordinated all research and revised the draft.