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Mutagenesis Advance Access originally published online on September 2, 2008
Mutagenesis 2008 23(6):501-507; doi:10.1093/mutage/gen043
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© The Author 2008. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.

In vivo assessment of DNA damage and protective effects of extracts from Miconia species using the comet assay and micronucleus test

Juliana Mara Serpeloni1,*, Mariana Bisarro dos Reis1, Juliana Rodrigues2, Lourdes Campaner dos Santos2, Wagner Vilegas2, Eliana A. Varanda3, Anne L. Dokkedal4 and Ilce Mara S. Cólus1

1Department of General Biology, Biological Sciences Center, Londrina State University, Londrina, Parana, Brazil 2Araraquara Institute of Chemistry, São Paulo State University, Araraquara, São Paulo, Brazil 3Department of Biological Sciences, Araraquara Faculty of Pharmaceutical Sciences, São Paulo State University, Araraquara, São Paulo, Brazil 4Department of Biological Sciences, Bauru Faculty of Sciences, Paulista State University, São Paulo, Brazil

The genus Miconia comprises ~1000 species belonging to the Melastomataceae family. Several crude plant extracts from Miconia and their isolated compounds have shown biological activities, such as analgesic and anti-neoplastic action; however, no studies concerning their effects on DNA are available. The present study aimed to evaluate, in vivo, the genotoxic and mutagenic effects of four species of plants from Miconia genus using the comet assay and micronucleus test. Their possible protective effects were also evaluated in experiments associating the plant extracts with cyclophosphamide (CPA). The methanolic extracts of Miconia albicans, Miconia cabucu, Miconia rubiginosa, Miconia stenostachya and the chloroformic extract of M. albicans were investigated. For genotoxic and mutagenic evaluations, three concentrations were tested, 200, 400 and 540 mg/kg body weight (bw), based on the solubility limit of the extract in distilled water. For the protective effects, only the highest dose was evaluated against 40 mg/kg bw of CPA. Blood was removed from mice tails pre- (T0) and post-treatment (T1–30 h) for the micronucleus test and 24 h post-treatment for the comet assay. The Student's t-test was used to compare data obtained at T0 and T1, the analysis of variance–Tukey test was used to compare between groups in the micronucleus test and the Kruskal–Wallis and Dunn's test were used to compare different groups in the comet assay. All the extracts induced alterations in DNA migration (comet assay); however, no mutagenic effect was observed in the micronucleus assay. All extracts showed a protective effect against CPA in both assays. Our study showed that the use of crude extracts could be more advantageous than the use of isolated compounds. The interaction between phytochemicals in the extracts showed efficacy in reducing mutagenicity and improving the protective effects.


   Introduction
Top
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
The use of plants for healing purposes is becoming increasingly popular, as they are believed to be beneficial and free from side effects. However, most of the information available on many medicinal herbs has no supporting scientific data and their use as medicaments is based solely on traditional folk usage that has been perpetuated down through the generations (1Go).

Miconia is a genus including ~1000 species occurring in tropical America (2Go,3Go). The genus belongs to the Melastomataceae family, which is pantropical with >166 genera that include ~4300 species (2Go). Many of these plants are used as medicine by people living in the Cerrado area, a Brazilian savannah ecosystem.

Studies aiming to describe the diverse biological activities of the Miconia species have shown promising results. Some studies have described the analgesic effects of crude extracts (hexane, methylene chloride and ethanol) obtained from Miconia species (4Go–6Go). Triterpenes ursolic acid, oleanolic acid and gypsogenic acid of Miconia fallax DC. and Miconia stenostachya (Schrank & Mart.) DC. were active against blood trypomastigote forms of Trypanosoma cruzi (7Go). Phenolic compounds from Miconia myriantha Benth showed inhibitory effects against aspartic proteases secreted by Candida albicans (8Go). Primin, an antibiotic extracted from Miconia sp. (Herb. I.A.-1903) with a 2-metoxi-6-n-pentil-p-benzoquinone structure, presented strong anti-neoplastic action in cases of patients presenting basal cell carcinoma and in one case of a patient presenting Kaposi's sarcoma (9Go).

Although numerous biological activities of the Miconia species have been reported, no studies concerning their effects on DNA (induction, reduction or mutation prevention) are currently available for the species. Mutations in somatic cells are important in the evolutionary process, but are also involved in the mechanisms of carcinogenesis and have a relevant role in degenerative chronic diseases, such as arteriosclerosis and heart diseases, which are the main causes of death in the human population (10Go).

Since most cancers are likely to be associated with mutagens and/or mitogens, research focusing on compounds that may inhibit or reverse either of the related processes may be crucial in the search for chemotherapeutic agents (11Go). Epidemiological studies have suggested that a reduced risk of cancer is associated with high consumption of vegetables and fruit. Thus, the cancer chemopreventive potential of naturally occurring phytochemicals is of great interest (12Go). Anti-mutagenic activity has been described for compounds isolated from plants, such as phenolic acids (ferulic and gentisic acids) (13Go), tannins and flavonoids (14Go–16Go) and terpenes (17Go).

The aim of the present study was to evaluate the genotoxic and mutagenic potential of extracts from four Miconia species in vivo, using the comet assay and the micronucleus test, as well as the possible protective effects of these extracts against cyclophosphamide (CPA)-induced DNA damage.


   Materials and methods
Top
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Chemicals
CPA (CAS: 50-18-0) was purchased from Sigma Chemical Co. (St. Louis, MO) and was diluted in distilled water and used as positive control and as the damage-inducing agent in the tests concerning possible protective effects.

Animals
Male and female albino Swiss mice (Mus musculus), aged 7–8 weeks old and weighing ~30 g at the beginning of the experiments, were used for the comet assay and micronucleus test. They were obtained from the mouse-breeding colony at the State University of Londrina (Paraná, Brazil) and were kept individually in polypropylene cages following the conditions for animal care recommended by the Canadian Council on Animal Care (18Go).

The mice (n = 250) were divided into groups of 10 (five males and five females) for each treatment and these (animals) mice were the same for both the end points (comet assay and micronucleus test).

Plant material and extract preparation
The aerial parts of Miconia cabucu Hoehne were collected in April 2005 at Pariquera-Açu, São Paulo State, Brazil, and authenticated by Dr Jorge Yoshio Tamashiro from the Instituto de Biologia, Universidade Estadual de Campinas, São Paulo. A voucher specimen (no. 1430) has been deposited in the Herbarium of the Universidade Estadual de Campinas, Brazil.

Miconia rubiginosa (Bonpl.) (voucher BOTU 25.376) and M. stenostachya DC (voucher BOTU 25.377) were collected in March 2005 at Palmeiras da Serra, Pratânia, São Paulo State, Brazil and authenticated by Luiz Fernando Rolim de Almeida from the Instituto de Botânica, Universidade Estadual Paulista (UNESP). Voucher specimens were deposited at the Herbarium ‘Irina Delanova Gemtchujnicov’ Instituto de Biociências, UNESP, Botucatu, São Paulo, Brazil.

Miconia albicans (Sw.) Steud leaves were collected in the UNESP campus of Bauru (São Paulo, Brazil) and identified by Dra. Anne L. Dokkedal. A voucher specimen was deposited in the Herbarium of the Biology Department of UNESP/Bauru, under number ALD 145.

The aerial parts obtained were dried (at 40°C for 4 days) and powdered. The dry powdered material was macerated three times with 2 litre of chloroform and methanol successively at room temperature and left for 48 h in the respective solvent. The solvents were filtered and evaporated at 35°C under reduced pressure providing CHCl3 and MeOH extracts. The quantity of extract obtained from all species studied in this work was determined as a percentage: 15.0 and 3.1 for MeOH and CHCl3 of M. albicans and 3.3, 9.3 and 14.6, respectively, for M. cabucu, M. rubiginosa and M. stenostachya.

Experimental design
The mice were treated with 0.1 ml of each of the solutions for every 10 g of body weight (bw) and they received water and food ad libitum throughout the treatment period. To evaluate the genotoxicity and mutagenicity of the extracts, the extracts were assessed at three different doses: 200, 400 and 540 mg/kg bw, via gavage. These doses were based on the solubility limit of the methanolic extract of M. albicans in distilled water. Thus, only one dose was tested for all the extracts and the results obtained for all the extracts at the same dose were compared. Given the low solubility of the CHCl3 extract, it had to be diluted in 8% Tween, since a dose of 540 mg/kg bw was unobtainable in distilled water.

A negative control group, positive control group and solvent control group were established for the treatment of the mice with distilled water, CPA [40 mg/kg bw intraperitoneally (i.p.)] and 8% Tween, respectively. To assess the anti-mutagenic activity of four Miconia species, CPA was administrated in a single i.p. dose 1 h after the plant extract was administered via gavage. Only the highest dose, 540 mg/kg bw, of each extract was evaluated regarding its protective effects.

Comet assay
The alkaline version of the comet assay was performed according to the guidelines proposed by Singh et al. (19Go), with a slight modification (20Go). Twenty-four hours after the treatment, 20 µl of heparinized periphery blood was mixed with 120 µl of 0.5% low-melting-temperature agarose in phosphate-buffered saline (PBS) and applied to microscope slides pre-coated with 1.5% normal-melting-temperature agarose in PBS. The slides were covered with a microscope coverslip and refrigerated for 5 min to gel. This was followed by immersion in ice-cold alkaline lysing solution [2.5 M NaCl, 10 mM Tris, 100 mM ethylenediaminetetraacetic acid (EDTA), 10% dimethyl sulphoxide, 1% Triton X-100, final pH 10.0] for at least 1 h. The slides were then incubated for 20 min in ice-cold electrophoresis solution (0.3 M NaOH, 1 mM EDTA, pH > 13), followed by electrophoresis at 25 V:300 mA (1.25 V/cm) for 25 min (21Go). After electrophoresis, the slides were then neutralized (Tris 0.4 M, pH 7.5) and stained with ethidium bromide (20 µg/ml). One hundred cells per mouse (50 cells analysed in each slide) were scored at 400x using a fluorescence microscope (Nikon-Brazil) with a blue (488 nm) excitation filter and yellow (515 nm) emission (barrier) filter. One scorer analysed the slides throughout the study and all the slides were coded. Quantification of DNA breakage was realized by visual scoring and the cells were classified into four categories representing different degrees of DNA damage, ranging from no visible migration (class 0, undamaged cells) to the maximum length comet [class 3, maximally damaged cells (DC)] (22Go).

The frequency of DC was obtained by totalling the number of undamaged (class 0) and DC from classes 1, 2 and 3 and dividing by the total number of cells analysed in each treatment. The total score for 100 nucleoids was obtained by multiplying the number of cells in each class by the damage class, according to the formula modified from Manoharan and Banerjee (23Go). Total score = (0 x n0) + (1 x n1) + (2 x n2) + (3 x n3), where n = number of cells in each class analysed. Thus, the total score could range from 0 (100 cells presenting no damage) to 300 (all cells presenting damage class 3).

The percentage reduction in the comet assay for the treatments with extracts showing anti-genotoxicity was calculated according to Manoharan and Banerjee (23Go) and Waters et al. (24Go) using the formula:

Formula
where A is the group of cells treated with CPA (positive control); B is the group of cells treated with the extracts plus CPA and C is the negative control.

The data were not homogeneous with regard to variance, so the Kruskal–Wallis test followed by Dunn's post hoc test was used to analyse the results.

Micronucleus test
The micronucleus test was performed on peripheral blood cells from the tail vein according to the protocol described by Hayashi et al. (25Go), which uses slides pre-stained with acridine orange (CAS: 494-38-2). The glass slides were heated to ~70°C on a hot plate and a 10-µl drop of an aqueous solution of the dye (1 mg/ml) was placed on each slide and spread evenly over the surface with the end of a second well-cleaned slide. Once dry, the slides were kept in the dark at room temperature for at least 24 h.

An internal control was established for each mouse by preparing a test slide with a drop of blood taken from its tail before the first treatment (time T0). Thirty hours after the treatment with the different compounds (T1), blood was obtained by perforating the caudal vein of the mouse with a needle and collecting 5 µl drops, each of which was placed at the centre of a pre-stained slide and covered with a coverslip (24 x 50 mm). These slides were then kept in the dark at –20°C for a minimum of 24 h before the cytological examination of the blood cells was performed.

The cell preparations were examined under a fluorescence microscope (Nikon) with a blue (488 nm) excitation filter and yellow (515 nm) emission (barrier) filter, using an immersion objective. One thousand reticulocytes per treated mouse were analysed and the proportion of micronucleated cells was counted.

The mean frequencies of micronucleated cells obtained for times T0 and T1 in each treatment group were compared by the Student t test (P < 0.05). The statistical ANOVA–Tukey test was used to calculate the means and standard deviations in order to compare the results obtained for the groups treated with extracts and the negative control group. The percentage reduction in the frequencies of micronucleated cells from the treatments with extracts that showed anti-mutagenicity was calculated according to Manoharan and Banerjee (23Go) and Waters et al. (24Go) as described above.


   Results
Top
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
The results of genotoxicity, mutagenicity and protective effects obtained for males and females were grouped to constitute Tables I, II, III and IV; no statistical difference occurred among the results obtained for these groups, demonstrating that gender differences do not modify the activity of the plant extract in these mice.

Table I shows the results of the comet assay for all three doses (200, 400 and 540 mg/kg bw) of each of the five plant extracts. The average values of the scores and frequency of DC obtained for all the doses and extracts evaluated were statistically different from the negative control (P < 0.001), indicating genotoxic activity.


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  		Table I. Detection of DNA damage using the comet assay of 100 white blood cells per mouse (n = 10 mice per group) exposed to water, 8% Tween, CPA and plant extracts of Miconia species and sampled at 24 h (mean ± SD)

 
Negative results for the micronucleus test were observed for all treatment groups (Table II), while statistically significant differences between T0 and T1 (Student t test) were detected only for the positive control group (CPA). After 30 h of treatment (T1), the data of all the experimental groups were compared with each other using the ANOVA and Tukey tests. Only the positive control group differed from the remaining groups (P < 0.001).


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  		Table II. Frequency of micronucleated reticulocytes for a total of 1000 analysed cells per mouse in each acute treatment to evaluate the mutagenicity of three different doses of the extracts of Miconia species and their positive and negative control groups

 
All extracts presented protective activity, since the results were statistically different from those obtained from the positive control group. Analysis of the values obtained for the extracts revealed a reduction in the scores and frequency of DC in relation to the group treated with CPA (P < 0.001) (Table III), as well as a reduction in the frequency of micronucleated cells (Table IV) (P < 0.001).


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  		Table III. Detection of DNA damage using the comet assay of 100 white blood cells per mouse (n = 10 mice per group) exposed to water, 8% Tween, CPA and vegetal extracts of Miconia species associated with CPA and sampled at 24 h (mean ± SD)

 


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  		Table IV. Frequency of micronucleated reticulocytes in a total of 1000 analysed cells per mouse in each acute treatment to evaluate the anti-mutagenicity of the extracts of Miconia species and their positive and negative control groups

 
The percentage reductions in DNA damage obtained in the comet and micronucleus assays after treatments with different extracts of Miconia species were calculated and are presented in Figure 1. The frequency of DC diminished by 41.33% under the treatments with the MeOH extract of M. albicans and M. stenostachya. The respective minimum and maximum reductions in scores observed for all species were 44.22% for M. rubiginosa and 49.90% for M. albicans. The reduction in micronucleated cells was more expressive (69.78%) in the group treated with the CHCl3 extract of M. albicans and less expressive (52.20%) for the MeOH extract of the same species.


Figure 1
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  		Fig. 1. Percentage of damage reduction promoted by the extracts in the presence of the CPA, calculated according to the formula of Manoharan and Banerjee (23Go) and Waters et al. (24Go). The parameters shown are the average score, frequency of DC and frequency of micronucleated reticulocytes (MNRET).

 

   Discussion
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 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
In order to develop unknown products and chemicals, it is necessary to determine their potential mutagenic effects. A balance between the therapeutic versus toxicological effects of a compound is important when verifying its applicability as a pharmacological drug (26Go).

All of the Miconia extracts evaluated presented low levels of genotoxicity according to the comet classes detected, with a predominance of comet class 1. The values obtained for cells treated with extracts were mid-range between those obtained in the negative control group and the group treated with the known mutagen CPA. This fact suggests moderate genotoxicity for these extracts. In the comet assay, it was possible to quantify and to distinguish cells with different rates of DNA damage, thus the analysis of the average values of the scores for each group was very important. In the present work, a predominance of comet class 1 was observed for all the treatment groups and almost no predominance for comet classes 2 and 3, differing from the data obtained for CPA, where comet classes 1 and 2 predominated.

Anomalies in the DNA molecule, such as adducts and single- and double-strand breaks, might cause lesions that become permanent and the use of the micronucleus test is an excellent means of evaluating any permanent damage in the genetic material. The same extracts used in the present study were evaluated by our group concerning their potential cytotoxicity (clonogenic assay) and mutagenicity (micronucleus test) in V 79 cell line in vitro and three non-cytotoxic concentrations, chosen through clonogenic assay (5, 10 and 20 µg/ml), showed absence of mutagenicity (Serpeloni et al., unpublished data).

The frequency of micronucleated cells was evaluated 30 h after the treatment. This time was chosen according to the literature data (27Go,28Go), which describe kinetics of micronucleus formation and cell migration. Both studies showed an elevation in the frequency of micronucleated cells even at 24 h and the peak of these cells in the peripheral blood is ~36 h after treatment. In the present study, no increase was observed 30 h after the treatment and all experimental groups showed the same frequency of micronucleated cells obtained in the negative control, which permitted the conclusion that the extracts were not mutagenic. Therefore, the negative results for mutagenicity observed in vivo in the present study may indicate that (i) the primary lesions (strand breaks) in the comet assay were repaired and this prevented the observation of micronuclei; (ii) the lesions induced in the comet assay were mainly single-strand breaks, which are less effective at producing chromosome fragments than double-strand breaks (29Go) and (iii) the prevalent DNA lesions leading to effects in the comet assay may have been DNA modifications, such as abasic sites involved predominantly in the induction of gene mutations (30Go) rather than in micronucleus induction.

The combination of these two assays in the present work proved to be both adequate and useful in the evaluation of the genotoxicity of the extracts from Miconia species, due to their complementary action.

CPA is an alkylating agent, which after administration is widely distributed throughout the body with a low degree of plasma protein binding (20%). Once activated, besides causing monoadducts, it can induce the formation of DNA–DNA and DNA–protein cross links (31Go) and generate free radicals that cause reactive species in a secondary mechanism leading to DNA damage (32Go). Consequently, CPA has been used as a clastogenic agent to induce cellular damage in the micronucleus test for evaluating mutagenic and anti-mutagenic activities (33Go).

In the present study, the protective effects of the Miconia extracts against CPA-induced DNA damage were observed via the comet assay and in the micronucleus assay. These effects can be explained as the result of the chemical constituents present in the extracts. A partial chemical analysis of the methanolic extract of M. cabucu showed that polyphenols (flavonoids, tannins and phenolic compounds) are the main compounds of this extract (34Go). Fractionation of the methanolic extract from M. cabucu was performed by Rodrigues et al. (34Go) and led to the isolation of the biflavonoid 5-hydroxy-4', 7-dimethoxyflavone-(6-C-6'')-5''-hydroxy-3''',4''',7''-trimethoxyflavone, gallic acid, flavonoids quercetin-3-o-β-xylopyranosyl-(1->2)-o-{alpha}-rhamnopyranoside, quercetin-3-o-{alpha}-rhamnopyranoside, myricetin-3-o-{alpha}-rhamnopyranoside, quercetin-3-o-β-glucopyranoside, kaempferol-3-o-β-(6''-coumaroyl)-glucopyranoside and myricetin-3-o-β-xylopyranosyl-(1->2)-o-{alpha}-rhamnopyranoside, all of them already reported in the literature.

The study of M. rubiginosa led to the identification of gallic acid, quercetin-3-o-{alpha}-rhamnopyranoside, quercetin-3-o-β-arabinofuranoside, quercetin-3-o-{alpha}-arabinopyranoside, quercetin-3-o-β-arabinopyranoside, quercetin-3-o-β-galactopyranoside, quercetin-3-o-{alpha}-rhamnopyranosil-(1->4)-o-β-galactopyranoside and epicatechin. The ongoing study of the methanolic extract of M. stenostachya has led to the isolation of glycosylated flavonoids derived from quercetin and myricetin and catechin derivates. Data from M. albicans are not yet available. The analysis of M. rubiginosa and M. stenostachya extracts (unpublished data) also showed that the extracts are chemically similar, justifying the similarities in the results observed in this study.

According to Scalbert and Williamson (35Go), most of the polyphenols ingested (75–99%) are not found in the urine and bioavailability studies have shown that maximum flavonoids concentrations are most often reached 1–2 h after ingestion, except for polyphenols, which are absorbed only after partial degradation by the colon microflora. According Anderson et al. (33Go), peak CPA concentrations in the blood plasma occur ~1 h after administration. Considering that the metabolism of CPA is faster than that of the extract components and the ample absorption of the same, the 1-h interval between the administration of the extracts and CPA injection aimed to place the CPA and extract metabolites in contact at the same time in the blood plasma.

A wide variety of botanical material, mostly dietary flavonoids and phenolic substances, have been reported to present expressive anti-carcinogenic and anti-mutagenic activities due to their antioxidant and anti-inflammatory properties (36Go). As antioxidants, flavonoids are capable of inhibiting the formation of free radicals and also of eliminating them by donating hydrogen atoms to these molecules and interrupting the chain reaction (37Go).

Phenolic compounds are found naturally in various agricultural products, such as coffee beans, fruits, vegetables, tobacco leaves, olive oils and wines (38Go). Caffeic acid, a well-studied phenolic compound, exhibits a cytoprotective effect on endothelial cells against oxidized low-density lipoprotein (39Go). It also inhibits the oxidation of lipoprotein exposed to ferrylmyoglobin and recycles {alpha}-tocopherol from {alpha}-tocopherol radicals (40Go). Similar to other phenolic acids (ferulic and gentisic acids), caffeic acid exhibited a protective effect against the genotoxicity of acridine orange and ofloxacin in Salmonella typhimurium (13Go).

Tannins are one such class of compounds that are suspected of possessing protective properties (14Go). Fedeli et al. (41Go) showed that tannins are capable of protecting against DNA breakage at low concentrations, while at high concentrations they could be genotoxic. Chen and Chung (42Go) showed the protective effect of tannins against mutagenicity induced by agents, which, like CPA, show indirect mutagenicity: benzidine, 4-aminobi-phenyl, 3,3'-4,4'-tetraaminobiphenyl and N,N-N',N'-tetramethylbenzidine. Several authors also have shown that ellagic acid, tannin detected in the chemical analyses of Miconia extracts, presents protective effects for different types of cells treated with different mutagenic agents (43Go,44Go).

The additive and synergistic effects of phytochemicals in fruit and vegetables have also been proposed as responsible for their potent antioxidant and anti-cancer activities (45Go). Among the distinguished compounds detected in the Miconia extracts are tannins, phenolic acids and flavonoids, which have been described as anti-mutagenic agents when assessed separately. Their interactions and possible synergistic effects may facilitate the protective effects observed.

The present study showed that the use of crude extracts can be more advantageous than the use of isolated compounds. The interaction between phytochemicals in the extracts showed efficacy in reducing mutagenicity and improving the protective effects.


   Funding
Top
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
The Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) to Biota-Fapesp Program (02/05503-6).


   Acknowledgments
 
The authors thank Conselho Nacional para o Desenvolvimento Científico e Tecnológico for a scientific initiation scholarship to M. B. Reis and grants to W.Vilegas, E.A. Varanda and I.M.S. Cólus. J.M. Serpeloni thanks Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior for a Master's scholarship.

Conflict of interest statement: None declared.


   Notes
 
* To whom correspondence should be addressed. Tel: +55 43 33714191; Fax: +55 43 33714527; Email: julianaserpeloni{at}yahoo.com.br


   References
Top
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received on March 30, 2008; revised on July 29, 2008; accepted on July 29, 2008.


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