Detection of Genotoxic,Cytotoxic,and Protective Activities of Eugenia dysenterica DC. (Myrtaceae) in Mice

Abstract

Eugenia dysenterica DC. (Myrtaceae), popularly known in Brazil as cagaiteira, is a widespread plant species in the Brazilian Cerrado. In folk medicine, the leaves of this plant are used to treat diarrhea and dysentery. The fruits are used for fresh consumption and industrial purposes. Because of the use of this plant as a therapeutic resource and food, the present study evaluated the genotoxic, cytotoxic, antigenotoxic, and anticytotoxic effects of the lyophilized ethanolic leaf extract of E. dysenterica using the mouse bone marrow micronucleus test. The genotoxicity and antigenotoxicity of this extract were evaluated using the frequency of micronucleated polychromatic erythrocytes, and the cytotoxicity and anticytotoxicity were assessed by the polychromatic and normochromatic erythrocyte ratio. According to our results, the lyophilized ethanolic leaf extract of E. dysenterica exhibited genotoxic and cytotoxic effects at the higher doses and protection against cyclophosphamide-induced genotoxic and cytotoxic actions at all doses tested.

Introduction

B razil is known for its rich biological diversity and also a number of plants that are used by the population in folk medicine to treat a variety of diseases.¹ Indeed, many plants are good sources of bioactive compounds that present health-promoting properties, such as antioxidant, anti-inflammatory, and antineoplastic activities.^2,3 However, many of them can also cause harm, including DNA damage, which may lead to genetic instability and related diseases, such as cancer.⁴ Therefore, plants exhibiting clear mutagenic properties should be considered as potentially unsafe, and they certainly require further testing before being recommended.⁵

Eugenia dysenterica DC., popularly known as cagaiteira in Brazil, is a medicinal plant of the Myrtaceae family with a wide range of uses in this country.⁶ The infusion prepared with E. dysenterica leaves is used in folk medicine to treat diarrhea and dysentery, whereas the cherry-like fruits are laxative.⁷ Moreover, cagaiteira fruits have shown economic importance because they are commonly consumed fresh or processed to obtain different types of sweets, popsicles, wine, and ice cream.^8,9

Analyses of E. dysenterica were carried out using several physical and chemical techniques, and its leaves were found to contain high levels of phenolic compounds, such as flavonoids and tannins, as well as the presence of saponins and terpenes.^10
–12 This plant species presents molluscicidal¹³ and antihelmintic^14,15 activities, suppresses fungal growth,¹¹ and induces toxicity in rats.^16,17 However, to the best of our knowledge, there are no reports on genotoxic and antigenotoxic effects of E. dysenterica.

In order to contribute to the safe and efficient use of this traditional medicinal plant as well as its bioactive compounds, in the present work we evaluated the genotoxic, cytotoxic, antigenotoxic, and anticytotoxic effects of lyophilized E. dysenterica ethanolic leaf extract (ELE) using the mouse bone marrow micronucleus (MN) test. This is a well-established assay that assesses MNs originating from chromosome fragments or whole chromosomes that are not included in the main nuclei during nuclear division. MN frequency in mouse bone marrow polychromatic erythrocytes (PCEs) is a very sensitive index of damage produced by ionizing radiation and chemical mutagens.¹⁸ Furthermore, the relationship between MN formation and potential carcinogenicity is well known.¹⁹

Materials and Methods

Plant material extraction

Leaves of E. dysenterica were collected in Senador Canedo in the state of Goiás, Brazil, in February 2005 and identified by Dr. Heleno Dias Ferreira (Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Goiás). A voucher specimen (number 283131/UFG) was deposited at the Herbarium of the Universidade Federal de Goiás. The leaves were dried at 45°C in a stove equipped with a forced ventilation system and exhaustively extracted with 96% aqueous ethanol (1 L) at room temperature (25°C) for 3 days. The alcohol solution obtained was filtered, lyophilized, transferred to glass flasks filled to the top, and kept at 5°C until the moment of use. Lyophilized E. dysenterica ELE was dissolved in water just before the use in the experiments and administered according to mouse body weight.

Experimental procedure

This protocol was approved by the Human and Animal Research Ethics Committee of the Universidade Federal de Goiás. Healthy, young male adult outbred mice (Mus musculus, Swiss Webster), obtained from the Central Animal House of the Universidade Federal de Goiás, were randomly allocated to treatment groups. All animals were brought to the laboratory 5 days before the experiments and housed in plastic cages (40 cm×30 cm×16 cm), in groups of five animals, in air-conditioned rooms at 22±2°C and 50±10% relative humidity, with a 12-h light–dark natural cycle. All the animals received food (an appropriate commercial rodent diet, Labina, from Ecibra Ltda.) and water ad libitum. On the day of dosing, the animals were approximately 7–9 weeks old and weighed 25–35 g.

For each treatment, groups of five animals were treated according to body weight with 50, 100, 150, or 200 mg/kg ELE injected intraperitoneally for the evaluation of genotoxicity. The same doses of ELE were administered in co-treatment with cyclophosphamide (CP) for the evaluation of antigenotoxicity. A positive (24 mg/kg i.p. CP) and a negative control group (distilled sterile H₂O) were included.

The animals were euthanized by cervical dislocation 24, 48, or 72 h after ELE administration, and their bone marrow cells were flushed from both femurs in fetal calf serum (lot number 30721063, Laborclin). After centrifugation (300 g, 5 min), the bone marrow cells were smeared on glass slides, coded for blind analysis, air-dried, and fixed with absolute methanol (CH₃OH) (lot number 55026, Synth) for 5 min at room temperature. The smears were stained with Giemsa (lot number 1081, Dole), dibasic sodium phosphate (Na₂HPO₄·12H₂O) (lot number 982162, Vetec), and monobasic sodium phosphate (NaH₂PO₄·H₂O) (lot number 983831, Vetec) for the detection of micronucleated PCEs (MNPCEs). For each animal, we prepared three slides and counted 2000 PCEs to determine the frequency of MNPCEs using microscopy (BH-2 microscope, Olympus) (10×100). Genotoxicity and antigenotoxicity were assessed by the frequency of MNPCEs, whereas cytotoxicity and anticytotoxicity were evaluated by the ratio of PCEs to normochromatic erythrocytes (NCEs). The MN test and MNPCE scoring were carried out according to the technique of Schmid.²⁰

Statistical analysis

In order to analyze ELE genotoxic activity, we compared the frequency of MNPCEs detected in the treated groups with the results obtained from the negative control group using one-way analysis of variance. To assess the antigenotoxic activity of ELE, the frequency of MNPCEs in the treated groups was compared with the results of the positive control group (by analysis of variance). Tukey's test was applied for a multiple comparison after the analysis of variance, and a value of P<.05 was taken as the criterion of statistical significance. To evaluate the cytotoxicity of ELE, the PCE/NCE ratio of all treated groups was compared with the results obtained from the negative control group. To evaluate ELE anticytotoxicity, the PCE/NCE ratio of all treated groups was compared with the results obtained from the positive control group. A nonparametric χ² test was applied to determine the statistical significance of the results, and a value of P<.05 was considered significant.

Results

The results obtained from the mouse bone marrow cells 24, 48, and 72 h after intraperitoneal administration of ELE or 48 h after intraperitoneal administration of ELE plus CP are shown in Table 1.

Table 1.

Frequency of Micronucleated Polychromatic Erythrocytes and Polychromatic and Normochromatic Erythrocyte Ratio Observed in Bone Marrow of Mice Treated with Different Concentrations of Lyophilized Ethanolic Leaf Extract of E. dysenterica and Co-Treated with Cyclophosphamide and Their Respective Controls

	MNPCEs/2,000 PCEs
Sampling time, treatment	Individual data	Mean±SD	PCE/NCE ratio
24 h
0 (C–) alone	2,3,1,3,2	2.2±0.83	1.04
ELE alone
50 mg/kg	3,1,2,3,4	2.6±1.14^a	0.94^a
100 mg/kg	2,1,3,5,3	2.8±1.4^a	0.88^b
150 mg/kg	3,3,5,7,4	4.4±1.67^b	0.74^b
200 mg/kg	7,8,5,5,6	6.2±1.3^b	0.68^b
0 (C+)+24 mg/kg CP	18,34,25,22,30	25.8±6.3	0.35^b
48 h
0 (C–) alone	1,4,2,1,2	2±1.22	0.97
ELE alone
50 mg/kg	1,2,3,3,3	2.4±0.89^a	0.97^a
100 mg/kg	2,3,4,4,3	3.2±0.83^a	0.85^b
150 mg/kg	3,4,5,8,7	5.4±2.07^b	0.68^b
200 mg/kg	5,4,6,8,7	6±1.58^b	0.56^b
0 (C+)+24 mg/kg CP	41,45,37,33,32	37.6±5.45	0.35
ELE+24 mg/kg CP
50 mg/kg	14,12,17,12,16	14.2±2.28^c	0.48^c
100 mg/kg	9,15,14,15,16	13.8±2.77^c	0.44^c
150 mg/kg	12,11,16,15,14	13.6±2.07^c	0.55^c
200 mg/kg	10,7,8,5,10	8±2.12^c	0.54^c
72 h
0 (C–) alone ELE alone	3,3,3,4,1	2.8±1.09	1.03
50 mg/kg	5,5,3,1,3	3.4±1.67^a	1.02^a
100 mg/kg	2,5,6,2,3	3.6±1.81^a	0.88^b
150 mg/kg	3,6,5,4,7	5±1.58^b	0.87^b
200 mg/kg	5,6,6,8,8	6.6±1.34^b	0.72^b
0 (C+)+24 mg/kg CP	18,25,21,18,23	21±3.08	0.75^b

All results were compared with the respective control group at the respective time.

No significant difference compared with the negative control (C–) group (P>.05).

Significant difference compared with the C– group (P<.05).

Significant difference compared with the positive control (C+) group (P<.05).

CP, cyclophosphamide; ELE, lyophilized ethanolic leaf extract of E. dysenterica; MNPCE, micronucleated polychromatic erythrocyte; PCE, polychromatic erythrocyte; PCE/NCE, polychromatic/normochromatic erythrocyte ratio.

In this study, the negative control group presented a low MNPCE value, as expected, and the positive control caused a significant increase in MNPCEs compared with the negative control (P<.05), confirming the sensitivity of the test.

During genotoxicity analysis, the groups that received 50, 100, 150, and 200 mg/kg ELE showed a mean of 2.6, 2.8, 4.4, and 6.2 MNPCEs (per 2000 PCEs), respectively, 24 h after intraperitoneal administration, whereas in the negative control group it was 2.2, demonstrating genotoxic activity of ELE at the doses of 150 and 200 mg/kg at 24 h (P<.05). In the cytotoxicity analysis, PCE/NCE ratios of the groups that received 50, 100, 150, and 200 mg/kg ELE were 0.94, 0.88, 0.74, and 0.68, respectively, 24 h after intraperitoneal administration, whereas in the negative control group it was 1.04, evidencing cytotoxicity at the doses of 100, 150, and 200 mg/kg ELE at 24 h (P<.05).

In the analysis of genotoxicity at 48 h, the groups that received 50, 100, 150, and 200 mg/kg ELE presented a mean of 2.4, 3.2, 5.4, and 6.0 MNPCEs (per 2000 PCEs), respectively, whereas in the negative control group it was 2.0, showing a significant increase in MNPCEs after administration of 150 and 200 mg/kg ELE compared with the negative control group (P<.05), which demonstrates its genotoxic activity at higher doses at 48 h. In relation to ELE cytotoxicity, PCE/NCE ratios were 0.97, 0.85, 0.68, and 0.56 in the groups that received 50, 100, 150, and 200 mg/kg ELE, respectively, whereas in the negative control group it was 0.97, evidencing cytotoxicity at the doses of 100, 150, and 200 mg/kg at 48 h (P<.05).

The antigenotoxicity and anticytotoxicity assays were performed for all treatments at 48 h because of the significant effects on genotoxicity and cytotoxicity of CP at this time of administration.

The protective effect of ELE against CP genotoxicity, evaluated at 48 h, was demonstrated because the mean MNPCE values (per 2000 PCEs) were 14.2, 13.8, 13.6, and 8.0 in the groups exposed to 50, 100, 150, and 200 mg/kg ELE plus CP, respectively, whereas in the positive control it was 37.6. These results show that ELE strongly modulated the genotoxic activity of CP, demonstrating its antigenotoxic effect (P<.05). In relation to the protection of ELE against CP cytotoxic action, also analyzed at the same time, PCE/NCE ratios were 0.48, 0.44, 0.55, and 0.54 in the groups exposed to 50, 100, 150, and 200 mg/kg ELE plus CP, respectively, whereas in the positive control group it was 0.35, demonstrating ELE anticytotoxicity at all doses (P<.05).

During genotoxicity analysis at 72 h, the groups treated with 50, 100, 150, and 200 mg/kg ELE presented mean MNPCE values (per 2000 PCEs) of 3.4, 3.6, 5.0, and 6.6, respectively, whereas in the control group it was 2.8, showing a significant increase in MNPCEs after administration of 150 and 200 mg/kg ELE compared with the negative control group (P<.05). In the analysis of ELE cytotoxic activity at 72 h, PCE/NCE ratios of the groups that received 50, 100, 150, and 200 mg/kg ELE were 1.02, 0.88, 0.87, and 0.72, whereas in the negative control group it was 1.03, indicating that higher doses of ELE exhibited cytotoxicity at this time (P<.05).

Discussion

E. dysenterica is a Brazilian plant species that is well known in folk medicine because its fruits are considered laxative and the leaves are used to treat diarrhea and dysentery.^7,17 It also has economic importance because its fruits are consumed fresh or processed to obtain different types of products.^8,9 Despite the widespread usage of this plant in Brazil, it remains unknown if it can really pose any risks to the people and/or protect them against the genotoxic action of some compounds. The purpose of this study was to evaluate the genotoxic, cytotoxic, antigenotoxic, and anticytotoxic activities of E. dysenterica ELE using the mouse bone marrow MN test.

The MN test detects genetic alterations arising from chromosomal damage and/or damage to the mitotic apparatus caused by clastogenic or aneugenic agents. As MNs are indicative of irreversible DNA loss, their frequency may be used as a mutation index.¹⁹ It is known that there is a positive correlation between increased frequency of MNs and the appearance of tumors in rodents and humans.^19
–21

The results of the genotoxic assessment of ELE showed a significant increase in MNPCE frequency at the doses of 150 and 200 mg/kg compared with the negative control group (P<.05) at all times analyzed (24, 48, and 72 h). This indicates that ELE exhibited genotoxic (clastogenic and/or aneugenic) effect on the PCEs in mouse bone marrow cells at higher doses. These data are alarming because the Brazilian population frequently uses tea made from E. dysenterica leaves to treat diarrhea.

The MN test used in this study also detects cytotoxic effects through the PCE/NCE ratio. When normal proliferation of bone marrow cells is affected by a toxic agent, the number of immature erythrocytes (PCEs) decreases in relation to that of mature erythrocytes (NCEs), leading to a decrease in the PCE/NCE ratio.¹⁹ Our results, at all times tested, indicated that ELE presented cytotoxicity at 100, 150, and 200 mg/kg because these doses caused significant reductions in the PCE/NCE ratio compared with the negative control group (P<.05).

The chemotherapeutic agent CP was used in the analysis of ELE's genotoxic and cytotoxic protective effects. CP causes genotoxicity when it interacts with DNA, producing DNA adducts and cross-links, which cause blockage of DNA replication and result in the cytotoxic action of this compound.²² To interact with DNA, CP must be metabolically activated by hepatic oxidases, and the positive control group used in this study showed that the maximal production of MN occurred 48 h after ELE administration. Therefore, the protective effects of ELE were evaluated at this time. Our results indicated that ELE protected mouse cells against the genotoxic and cytotoxic activities of CP at all doses analyzed (50, 100, 150, and 200 mg/kg), which suggests the presence of antigenotoxic and anticytotoxic compounds in this plant species (P<.05).

Studies on the constituents of E. dysenterica leaves indicated that tannins and flavonoids, which are phenolic compounds, represent the major constituents.¹² Phenolic compounds are responsible for the prevention of diseases associated with oxidative damage of membranes, proteins, and DNA²³ and also present antimutagenic, anticarcinogenic, and anti-aging properties.²⁴ Previous studies showed a positive correlation between the antioxidant capacity of E. dysenterica and its total phenolic content using experiments with 1,1-diphenyl-2-picrylhydrazyl radical scavenging capacity and oxygen radical absorbance capacity.^7,25 Thus, it is possible that the common mechanisms of action of E. dysenterica flavonoids, such as antioxidant activity and free radical scavenging, could contribute to its antimutagenic profile.

Flavonoids exert biological and pharmacological effects arising mainly from their antioxidant properties,^24,26 cause apoptosis in cancer cells,^27,28 and present antitumor activity due to their ability to inhibit topoisomerase I and II.^29,30 However, it was reported that flavonoids can be either a protector or a cytotoxic agent.³¹ For example, quercetin, a flavonoid identified in E. dysenterica,²⁵ demonstrated protective and cytotoxic effects dose-dependently.³¹

Tannins are also cytotoxic compounds with antiviral and antibacterial activities.²⁴ It was reported that E. dysenterica presents toxicity in a variety of organisms.^{7,11,13

–17} Lima et al.¹⁷ studied the effects of ethanolic extract, aqueous extract, and infusion of E. dysenterica leaves on intestinal motility and antidiarrheal activity in animal models. The authors demonstrated that only the ELE had antidiarrheal activity, but the animals treated with this type of extract as well as with infusion presented severe damage to the intestinal tissue and liver.

In the present study, the protection against CP's genotoxic and cytotoxic actions can be attributed, at least partially, to the phenolic compounds of E. dysenterica, especially flavonoids and tannins, which are the major secondary constituents of this plant. Also, the cytotoxic and mutagenic effects found in this study can be attributed to the phenolic compounds present in this plant leaf extract.

Although phenolic compounds attenuate oxidative stress, the concerns over its toxicity have increased the interest in investigating their role in medicinal applications.³² In fact, most phenolic compounds that occur naturally present antioxidant effects.³³ However, depending on the redox potential, antioxidants can either accept or donate electrons, which may induce contradictory effects.³⁴ Reactive oxygen species produced by phenolic compounds can damage DNA³⁵ and cause mutagenesis in mammalian and bacterial systems, aging, and carcinogenesis, as well as have an antimicrobial effect.^26,36,37

Therefore, the cytotoxicity and genotoxicity as well as the modulated effects of genotoxicity and cytotoxicity detected in our experiments are in accordance with the study carried out by Hodek et al.²⁴ using phenolic compounds, which also presented contradictory activities. As already known, the final response of a treatment with a plant extract is the result of synergistic, antagonistic, and other interactive effects among its biologically active components.

The results presented in this study are extremely important because traditional medicine can help improve the development of more effective drugs with minimal or no side effects. Although E. dysenterica represents a potential source for the production of an antidiarrheal drug, our data indicated that at higher doses this plant can cause toxicity and mutations, important early factors in carcinogenesis. Therefore, based on our results, the usage of this plant in folk medicine should be viewed with caution.

Conclusions

In summary, our results indicated that the lyophilized ELE of E. dysenterica (a) presented genotoxic and cytotoxic activities at higher doses and (b) protected mouse cells against CP's genotoxic and cytotoxic activities at all doses analyzed.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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