Evaluation of the mutagenicity and antimutagenicity of Ziziphus joazeiro Mart. bark in the micronucleus assay.

The aim of this study was to evaluate the mutagenicity (clastogenicity/aneugenicity) of a glycolic extract of Ziziphus joazeiro bark (GEZJ) by the micronucleus assay in mice bone marrow. Antimutagenic activity was also assessed using treatments associated with GEZJ and doxorubicin (DXR). Mice were evaluated 24–48 h after exposure to positive (N-nitroso-N-ethylurea, NEU - 50 mg.kg−1 and DXR - 5 mg.kg−1) and negative (150 mM NaCl) controls, as well as treatment with GEZJ (0.5–2 g.kg−1), GEZJ (2 g.kg−1) + NEU and GEZJ (2 g.kg−1) + DXR. There were no significant differences in the frequencies of micronucleated polychromatic erythrocytes in mice treated with GEJZ and GEJZ + DXR compared to the negative controls, indicating that GEZJ was not mutagenic. Analysis of the polychromatic:normochromatic erythrocyte ratio revealed significant differences in the responses to doses of 0.5 g.kg−1 and 1–2 g.kg−1 and the positive control (NEU). These results indicated no systemic toxicity and moderate toxicity at lower and higher doses of GEZJ. The lack of mutagenicity and systemic toxicity in the antimutagenic assays, especially for treatment with GEZJ + DXR, suggested that phytochemical compounds in Z. joazeiro bark attenuated DXR-induced mutagenicity and the moderate systemic toxicity of a high dose of Z. joazeiro bark (2 g.kg−1). Further studies on the genotoxicity of Z. joazeiro extracts are necessary to establish the possible health risk in humans and to determine the potential as a chemopreventive agent for therapeutic use.

Biologically active compounds have been recognized for their pharmacological properties, but many of them are of limited therapeutic use because of their toxicological, carcinogenic and mutagenic properties (Ames, 1983;Konstantoupoulou et al., 1992;Tavares, 1996). The analysis of genotoxicity is a major aspect of drug development since most pharmaceutical companies evaluate the potential of a new therapeutic agent based on its genotoxicity in vitro and in vivo (Purves et al., 1995). In this context, the screening of popularly used plants and their isolated components for mutagenic activity is necessary and important for establishing adequate control measures. This screening can also provide insights into the mechanisms involved in the biological effects of plants used as therapeutic agents (Varanda, 2006).
As far as genotoxicity studies are concerned, the in vivo micronucleus (MN) assay in rodent bone marrow is a crucial part of the battery of tests used to identify hazardous mutagens (Mateuca et al., 2006). This assay is especially suited for assessing mutagenic hazards because it contemplates various factors, such as in vivo metabolism, pharmacokinetics and DNA repair mechanisms, even though these processes vary among species and tissues and have different genetic endpoints (OECD, 1997a,b;Ribeiro et al., 2003). Since bone marrow erythroblasts develop into polychromatic erythrocytes (PCEs), i.e., cells generated by extrusion of the main nucleus, micronuclei may remain in an otherwise anucleated cytoplasm. Consequently, the frequency of micronucleated polychromatic erythrocytes (MNPCEs) has been the principal endpoint for MN assays. The measurement of MNPCEs in peripheral blood is possible in any species in which the spleen does not remove micronucleated erythrocytes, or that is sufficiently sensitive to agents that cause structural or numerical chromosomal aberrations. An increase in the frequency of MNPCEs in treated animals, i.e., a positive result, indicates that a substance can cause the formation of micronuclei through chromosomal damage or damage to the mitotic apparatus of erythroblasts. On the other hand, a negative result implies that the test substance does not cause micronucleus formation in immature erythrocytes. The number of normochromatic erythrocytes (NCEs) in peripheral blood that contain micronuclei for a given number of mature erythrocytes can also be used as the endpoint of this assay (OECD, 1997c;Ribeiro et al., 2003). Several studies have used the mammalian in vivo MN assay to understand the mutagenic effects induced by phytotherapeutics and foods (Indart et al., 2007;Venkatesh et al., 2007;Chandrasekaran et al., 2011;Silva et al., 2011;Alves et al., 2012).
Although several studies have examined the potential therapeutic effectiveness of Z. joazeiro Mart., there has been no systematic investigation of the genotoxic and mutagenic effects of this plant. In this work, we examined the mutagenic effects of a glycolic extract of Z. joazeiro Mart. bark as part of a wider study on the genotoxic potential of herbal extracts. The effect of the maximum permissible concentration of Z. joazeiro Mart. on the mutagenicity of doxorubicin (DXR) in mouse bone marrow, i.e., its antimutagenic activity, was also examined.

Raw material and sample preparation
A glycolic extract of Z. joazeiro bark (GEZJ) was purchased commercially and stored according to the manufacturer's recommendations (AKSY Comercial Ltda., São Bernardo do Campo, SP, Brazil). Aliquots (1.5 L) of this extract were submitted to solvent removal proceedings by rotary evaporation (40 rpm) (Rotavapor model R-215) coupled to a bath heating system maintained at 50-60°C (Bath Heating model B-491), a vacuum pump (vacuum of 500 mm Hg; Vacuum Pump V-700 with Automatic Vacuum Controller V-855), a water recirculator (Recirculator Chiller F-100) and an evaporation bottle (Büchi Labortechnik AG, Switzerland). The final product was transferred to a 1 L reaction bottle (SCHOTT ® DURAN ® ) and kept at -20°C for 24 h in order to evaluate the freezing of the final product and the efficacy of solvent evaporation (Agência Nacional de Vigilância Sanitária (ANVISA) (2010)). Aliquots (40 mL) of this final product were transferred to penicillin-type glass vials (50 mL) and lyophilized (Lyophilizer model Alpha 1-2 LDPlus, Martin Christ Gefriertrocknungsanlagen GmbH, Germany) and the dry mass were measured (Electronic Analytical Balance AUW-220D, Shimadzu Corp., Kyoto, Japan). Aqueous solutions of the lyophilized product were prepared in type 1 water at twice the final concentration, sterilized by filtration (Millipore Corporation, hydrophilic Durapore ® PVDF, 0.22 mm, ± 47 mm, cat. no. GVWP 047 00) and stored in sterile polypropylene tubes (50 mL) at -70°C until used.

In vivo assays
Healthy, heterogeneous, young adult male and female Swiss mice (Unib:SW) 7-12 weeks old (pubescent period) weighing 30-40 g (weight variation among mice of each sex was < 20% of the mean weight) were provided by CEMIB (Centro Multidisciplinar para Investigação Biológica -UNICAMP) and erythrocytes from the bone marrow of these mice were used in the MN assay (Collaborative Study Group for the Micronucleus Test (CSGMT), 1986; Chorilli et al., 2007).
Animals of the same sex were housed in polypropylene boxes in an air-conditioned environment to 22 ± 3°C, with a relative air humidity of 50% ± 20% and a 12 h light/dark cycle. The mice were fed commercial rodent chow (Purina ® Labina, Nestlé Purina Pet Care Company) and water ad libitum, and were acclimated to laboratory conditions for seven days prior to use in the experiments. At the end of this period, each mouse was weighed and then received 2 mL of liquid (containing the desired test agent) per 100 g body weight.
All animals were properly identified by numerical markings on their tails to ensure continuity of the records and reliable interpretation of the results throughout the study (OECD, 1997c). After the period of treatment, the mice were euthanized by inhalation of carbon dioxide in adapted acrylic chambers as described in the Report of the American Veterinary Medical Association panel on euthanasia (Beaver et al., 2000). This study was done in accordance with the Universal Declaration of Animal Rights (UNESCO, 1978), the ethical principles for animal experimentation established by the Brazilian Society of Labora-tory Animal Science (SBCAL -Sociedade Brasileira de Ciência em Animais de Laboratório), the Brazilian Environmental Crimes Law (Law no. 9.605, February 12, 1998), the Brazilian standards for Didactic-Scientific Practice of Vivisection of Animals (Law no. 6.638, May 8, 1979), and was approved by the Committee for Ethics in Research Involving Animals at UNIFENAS (CEPEAU Protocol no. 04A/2008).

Processing of bone marrow
MN assays using bone marrow erythrocytes were done 24 h and 48 h after treatment, using previously described methodology (Schmid, 1976;Zambrano et al., 430 Micronucleus test of Z. joazeiro barks 1982). Shortly after euthanasia, the femora were surgically and aseptically removed and the mice were appropriately discarded. Each femur was sectioned at the proximal end and the contents of the spinal canal were washed with 1.5 mL of 150 mM NaCl and transferred to a 15 mL centrifuge tube. This material was resuspended with a Pasteur pipette to ensure a homogenous distribution of bone marrow cells. The suspension was then centrifuged at 1,000 rpm (Bench centrifuge, model NT 810, Nova Técnica Ind. e Com. de Equip. para Laboratório Ltda., Piracicaba, SP, Brazil) for 5 min. The supernatant was discarded and the resulting pellet was resuspended in 500 mL of 150 mM NaCI solution added 4% formaldehyde. The slides (two per animal) were prepared by smearing, dried at room temperature for 24 h and stained with Leishman's eosin methylene blue dye [pure dye for 3 min followed by diluted dye in distilled water (1:6) for 15 min] to differentiate polychromatic erythrocytes (PCEs) from monochromatic erythrocytes (NCEs).
PCEs were observed by light microscopy (Nikon Eclipse E-200 microscope) at a magnification of 1000x, counted (at least 2000 anucleated polychromatic erythrocytes per animal) with the aid of a digital cell counter (Contador Diferencial CCS02, Kacil Indústria e Comércio Ltda., PE, Brazil) and photographed using an 8.1 Megapixel Digital Camera (DC FWL 150). The number of PCEs, the number and frequency of MNPCEs and the ratio of polychromatic to monochromatic erythrocytes (PCE/NCE) were determined.

Statistical analysis
The data from the MN assay were analyzed by oneway analysis of variance (ANOVA) using a 9 x 2 x 2 (treatment x gender x time) factorial scheme followed by multiple comparisons with the Tukey test (a = 0.05). All analyses were done using SAS ® version 9.2 computer software.
phytochemical compounds responsible for the moderate toxicity (altered PCE/NCE ratio) of GEZJ (2 g.kg -1 ) in bone marrow may also have an important role in attenuating the mutagenicity (n and % of MNPCE) of DRX (5 mg.kg -1 ). The acute toxicity of different plant extracts, including Z. joazeiro, has previously been based on doses (1 to 4 or 5 g/kg) administered orally to different groups of mice (one dose per mouse, with each group containing eight animals: four males and four females) (Alviano et al., 2008). Behavioral parameters, including convulsion, hyperactivity, sedation, grooming, loss of righting reflex, increased or decreased respiration, and changes in food and water intake were also noted. These animals were observed and weighed over a period of 14 days; no weight loss was detected. Treated mice showed no behavioral alterations and the extract LD 50 values ranged from 2.0-3.5 g/kg. None of the extracts was lethal to mice at the doses tested and the data from the in vivo assays indicated that the extracts had low toxicity (Alviano et al., 2008). The data from the MN assays presented here provides additional information on the systemic toxicity of Z. joazeiro in mouse bone marrow based on the PCE/NCE ratio that suggested moderate toxicity of GEZJ at doses of 1.5-2 g.kg -1 that was independent of mouse gender but varied with time (24-48 h).
The PCE/NCE ratio is an indicator of the acceleration or inhibition of erythropoiesis and varies with the scoring interval. A continuous decline in the PCE/NCE ratio may reflect the inhibition of cell division, the killing of erythroblasts, the removal of damaged cells, or dilution of the existing cell pool with newly formed cells (Venkatesh et al., 2007). Several mechanisms may contribute to the cytotoxicity of DXR and MN induction (Gewirtz, 1999), including the intercalation of DXR in cellular DNA (Painter, 1978;Kiyomiya et al., 2001), stabilization of the topoisomerase II-DNA complex (Pommier et al., 1985;Guano et al., 1999), free radical-mediated toxicity caused by redox cycling of the semiquinone radical (Bachur et al., 1979), or the formation of reactive oxygen species by the DXR-iron complex (Eliot et al., 1984;Myers, 1998;Konorev et al., 1999). On the other hand, chemicals such as captopril and desferrioxamine (Al-Harbi, 1993;Al-Shabanah, 1993), bcarotene and vitamins A, C and E (Lu et al., 1996;Gulkac et al., 2004;Costa and Nepomuceno, 2006), thiol N-acetylcysteine, probucol, lovastatin and hydrophilic flavonoids such as rutin and luteolin (Al-Gharably, 1996;Sadzuka et al., 1997;D'Agostini et al., 1998;Bardeleben et al., 2002) can also reduce DXR-induced MN formation, genotoxicity and cytotoxicity. However, proponents of herbal medicine always claim that mixtures are better than pure chemicals because the dozens of biologically active compounds in plants work together to produce a greater effect than any one chemical on its own (Mackenzie, 2001).
Screening for newer pharmacological agents that can protect normal cells against DXR-induced cumulative toxicity is essential. Many plants widely used in traditional medicine are less toxic than pharmaceutical agents and have recently attracted the attention of researchers around the world. Plants contain many compounds and it is likely that these can provide better protection than a single molecule (Vidhya and Devraj, 1999). The presence of many molecules in plants may be advantageous, as some of them may counteract the toxicity of others so that the net effect may be therapeutically beneficial. For example, the effect of various concentrations (200,250,300,350 and 400 mg/kg body weight) of Aegle marmelos on DXR-induced mutagenicity in mouse bone marrow was studied (Venkatesh et al., 2007). Mice treated with different concentrations of DXR (5, 10 or 15 mg.kg -1 body weight) showed a dose-dependent elevation in the frequency of PCE and NCE in their bone marrow, and this was accompanied by a DXR-mediated dose-dependent decline in the PCE/NCE ratio. In contrast, the treatment of mice with A. marmelos orally once a day for five consecutive days before treatment with DXR significantly reduced the frequency of DXRinduced micronuclei and significantly increased the PCE/NCE ratio at all time intervals. This chemoprotective effect may reflect the sum of interactions between different components of this complex mixture. The degree of protection may depend on the individual or collective interaction of components with the genotoxic agent. The plausible mechanisms of action of A. marmelos in protecting against DXR-induced damage included the scavenging of O 2 •-, • OH and other free radicals, an increase in antioxidant status, restoration of topoisomerase II activity and inhibition of the formation of the DXR-iron complex (Venkatesh et al., 2007). More recently, Alves et al. (2012) evaluated the genotoxic potential of a hydroalcoholic extract of Copaifera lansdorffii Desf. leaves and its influence on the genotoxicity of DXR (MN test) in peripheral blood from Swiss mice. Their finidngs demonstrated that C. lansdorffii Desf. was not genotoxic but that the extract significantly reduced the number of micronuclei in DXR-treated mice. The putative antioxidant activity of one or more of the active compounds of C. lansdorffii Desf., including two major flavonoid heterosides (quercitrin and afzelin), may explain the effect of this plant on DXR genotoxicity.

Conclusions
This study used the MN assay to evaluate the mutagenic (clastogeny and/or aneugeny) and antimutagenic activity of an extract of Z. joazeiro bark in mouse bone marrow. The Z. joazeiro bark extract was not mutagenic at the doses and time intervals tested, although sex-related variation was observed. The antimutagenic effect (anticlastogeny and/or antianeugeny) of Z. joazeiro bark extract against DXR-induced genotoxicity was observed at a high dose of extract (2 g.kg -1 ), but was independent of the duration of treatment and animal sex. Low concentrations of GEZJ (0.5-1 g.kg -1 ) were not toxic, regardless of mouse gender and duration of treatment, whereas moderate toxic-ity was observed at doses of 1.5-2 g.kg -1 . Together, these findings indicate that phytochemical compounds in Z. joazeiro bark can attenuate DRX-induced mutagenicity and that a high dose of extract (2 g.kg -1 ) showed no toxicity in the conditions tested here.
Other studies on the genotoxicity and mutagenicity of Z. joazeiro extracts are needed to characterize the (anti)genotoxic effects and mechanisms, and to determine the potential health risks of this extract in humans. Such investigations will be useful for implementing strategies related to the use of Z. joazeiro bark in chemoprevention.