Evaluation of the in Vivo Mutagenic Potential of Hydroalcoholic Extracts of the Northern Highbush Blueberry (Vaccinium Corymbosum L
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Genetics and Molecular Biology, 31, 2, 555-560 (2008) Copyright © 2008, Sociedade Brasileira de Genética. Printed in Brazil www.sbg.org.br Research Article Evaluation of the in vivo mutagenic potential of hydroalcoholic extracts of the northern highbush blueberry (Vaccinium corymbosum L. Ericales, Ericaceae) on peripheral blood cells of Swiss mice (Mus musculus Rodentia, Muridae) Patrícia Scotini Freitas1, Sérgio Faloni de Andrade2 and Edson Luis Maistro3 1Faculdade de Enfermagem, Universidade José do Rosário Vellano, Poços de Caldas, MG, Brazil. 2Núcleo de Investigações Químico-Farmacêuticas, Universidade do Vale do Itajaí, Itajaí, SC, Brazil. 3Departamento de Fonoaudiologia, Faculdade de Filosofia e Ciências, Universidade Estadual Paulista, Marília, SP, Brazil. Abstract The northern highbush blueberry (Vaccinium corymbosum L. Ericales, Ericaceae) is very rich in anthocyanins, natu- ral pigments which have strong antioxidant properties and potential health benefits, resulting in the worldwide use the blueberry as a medicinal plant. We investigated the mutagenic potential of simple hydroalcoholic extracts of V. corymbosum acutely administrated by gavage to Swiss mice at doses of 1 g kg-1, 1.5 g kg-1 and 2 g kg-1. Peripheral blood cells were collected 4 h and 24 h post-gavage and assessed by the alkaline comet assay, with further blood samples being collected at 48 h and 72 h for assessment using the micronucleus (MN) assay. Our results show that the V. corymbosum extracts did not induce any statistically significant increase in the average amount of DNA dam- age in peripheral blood leukocytes. However, we did record a significant increase in the frequency of micronucleated polychromatic erythrocytes at the three doses tested. Key words: comet assay, micronucleus, mutagenicity, Vaccinium corymbosum. Received: June 20, 2007; Accepted: September 13, 2007. Introduction nins, which includes antioxidant activity (Wang et al., The medicinal use of plants is of great antiquity, with 1997; Mazza et al., 2002; Lohachoompol et al., 2004), current records showing that more than 150,000 plant spe- anti-inflammatory effects (Youdim et al., 2002), cardiovas- cies have been studied for their medicinal properties. Many cular protection, antidiabetic properties, improvements to plants contain therapeutic substances (Hoyos et al., 1992; vision and inhibition of carcinogenesis (Cabrita and An- Surh and Ferguson, 2003) which can be extracted and used dersen, 1999; Camire, 2000; Katsube et al., 2003). The in the preparation of medicines, or the plant itself can be fruits of the economically important northern highbush used directly as a medication, a practice that is particularly blueberry (Vaccinium corymbosum L.) are consumed in popular in developing countries. However, many plants many countries throughout the world because they contain contain compounds which are known to cause ill-health, or large amounts of antioxidants thought to be beneficial to even death, in animals and humans, there is considerable health (Prior et al., 1998; Ehlenfeldt and Prior, 2001). interest in determining the health risks associated with We assessed the in vivo mutagenic potential of three plants used for medicinal purposes. different concentrations of simple hydroalcoholic extracts The genus Vaccinium (Ericales, Ericaceae) comprises of V. corymbosum using Swiss mice peripheral blood cells about 200 species, some of which produce edible fruits of subjected to the alkaline comet assay and the micronucleus economic importance. In recent decades interest in the (MN) assay. anthocyanin content of some Vaccinium species has re- vived due to the pharmacological properties of anthocya- Material and Methods Send correspondence to Edson Luis Maistro. Departamento de Plant material and chemicals Fonoaudiologia, Faculdade de Filosofia e Ciências, Universidade Estadual Paulista, Av. Hygino Muzzi Filho 737, Caixa Postal 181, Northern highbush blueberry (Vaccinium 17525-900 Marília, SP, Brazil. E-mail: edson.maistro@marilia. corymbosum L. Ericales, Ericaceae) berries were collected unesp.br. in an experimental field in the town of Videira 556 Freitas et al. (27°00'04.76" S, 51°07'29.77" W), the field belonging to fications introduced by Klaude et al. (1996) as well as some the Agricultural Research and Extension Education Com- additional modifications. Briefly, for each mouse periph- pany (Pesquisa Agropecuária e Extensão Rural de Santa eral blood was collected from the orbital vein at 4 h and 24 h Catarina S.A., EPAGRI) in the southern Brazilian State of post-gavage and a 10 μL aliquot mixed with 120 μLof Santa Catarina. The berries were picked in October 2005 at 0.5% (w/v) LMP agarose at 37 °C and rapidly spread on a the commercially ripe stage and all damaged, diseased or microscope slide pre-coated with 1.5% (w/v) normal melt- pest-infested fruits, stems or leaves were removed. The ber- ing point agarose. Coverslips were added and the slides ries were kept in polyethylene bags at -20 °C until extract were allowed to gel at 4 °C, for 20 min before gently re- preparation. Prior to extraction, the frozen berries were moving the coverslips and immersing the slides in cold crushed using a food processor and 1 kg of berries macer- freshly-prepared lysing solution consisting of 89 mL of a ated with 70% (v/v) aqueous ethanol at room temperature stock solution (890 mL of distilled water containing 2.5 M (22 °C) for seven days. The simple hydroethanolic extract NaCl, 100 mM EDTA, 10 mM TRIS and 1% (w/v) sodium was obtained by filtration and concentration of the filtrate lauryl sarcosine with the pH set to 10.0 by the addition of under reduced pressure using a rotary evaporator. The yield about8gofsolid NaOH) plus 1 mL of Triton X-100 and was 138 g, or 13.8% (w/w) with reference to the original 10 mL of DMSO. After treatment the slides to stand at 4 °C weight of the berries. for 1 h protected from light and then placed at the anode of a The principal chemicals used were obtained from the gel electrophoreses box containing high pH (> 13) electro- following suppliers: dimethyl sulphoxide (DMSO, Merck, phoresis buffer (300 mM NaOH per 1 mM EDTA, prepared CAS No. 67-68-5); ethidium bromide (Sigma, CAS No. from a stock solution of 10 N NaOH and 200 mM EDTA, 1239-45-8); ethylenediaminetetraacetic acid (EDTA, pH 10) at 4 °C for 20 min before electrophoresis to allow Merck); low melting point (LMP) agarose (Invitrogen the DNA to unwind. Electrophoresis was performed at 25 V 15517-014:); N-nitroso-N-ethylurea (ENU, Sigma, CAS and 300 mA for 20 min at 4 °C in an ice bath, after which n. 759-73-9); normal melting point (NMP) agarose (Invi- the slides were submerged in neutralization buffer (0.4 M trogen 15510-019); sodium N-lauroylsarcosine (Sigma L- Tris-HCl, pH 7.5) for 15 min, dried at room temperature 5125:) and tris(hydroxymethyl)aminomethane (TRIS, and fixed in 100% ethyl alcohol for 10 min. The slides were Merck, CAS No. 77-86-1) and Triton X-100 (Merck). dried and stored at least overnight before staining by cover- ing the preparation with 30 μL of 1x ethidium bromide Animals and assay procedures staining solution (prepared from a 10x stock solution con- We used 12-week old male and female Swiss mice taining 200 μgmL-1 ethidium bromide) and then covered (Mus musculus) with a body weight (bw) of 25 g to 30 g with a coverslip. The material was evaluated immediately which had been acquired from the animal house of the Jose at 400x magnification using a Nikon fluorescence micro- do Rosario Vellano University (UNIFENAS), Alfenas, scope with a 515 nm to 560 nm excitation filter and a Minas Gerais, Brazil. The mice were kept in polyethylene 590 nm barrier filter. For each mouse, the extent and distri- boxes in groups of six mice (three females and three males) bution of DNA damage indicated by the comet assay was in a climate-controlled environment (25 ± 4°C55± 5% hu- evaluated by examining at least 100 randomly selected and midity) with a 12 h (0700 to 1900) day-length. All the mice non-overlapping cells on the slides. The cells were visually had ad libitum access to water and a commercial laboratory scored, according to tail size, into four classes: (1) class 0, rat food (Labina: Purina Co., Brazil), and were euthanized undamaged and with no tail; (2) class 1, tail shorter than the 72 h after the respective treatments. The UNIFENAS Ani- diameter of the head (nucleus); (3) class 2, tail length 1 to 2 mal Bioethical Committee approved this study under proto- times the head diameter; and (4) class 3, tail longer than col number 03A/2006 in accordance with the Federal twice the head diameter. Comets with no heads and images Government legislation on animal care. with nearly all DNA in the tail, or with a very wide tail, The V. corymbosum extract was administered to each were excluded from evaluation because they probably rep- mouse as a single 0.5 mL gavage dose adjusted to contain resent dead cells (Hartmann and Speit, 1997). The total extract equivalent to1gkg-1 bw, 1.5 g bw kg-1 or2gkg-1 score for 100 comets was obtained by multiplying the num- bw, this range being based on a previous Swiss mice acute ber of cells in each class by the damage class, ranging from toxicity study involving doses of extract in excess of the 0 (all undamaged) to 300 (all maximally damaged). equivalent of2gkg-1 bw (data not shown). The negative The micronucleus (MN) assay followed the general control group received distilled water by gavage. The posi- protocol recommended by Krishna and Hayashi (2000) tive control group received 50 mg of N-nitroso-N-ethyl- with slight modifications. At 48 h and 72 h post-gavage pe- urea/kg (ENU) dissolved in pH 6 phosphate buffer and ripheral blood was collected from the orbital vein of the administered as a single intraperitoneal injection.