Protective effects of solvent fractions of Mentha spicata (L.) leaves evaluated on 4-nitroquinoline-1-oxide induced chromosome damage and apoptosis in mouse bone marrow cells

Spearmint leaves (Mentha spicata L.) contain high levels of antioxidants that are known to protect against both exogenous and endogenous DNA damage. In this study, the protective effects of the hexane fraction (HF), chloroform fraction (CF) and ethyl acetate fraction (EAF) in an ethanol extract from M. spicata were evaluated against 4-nitroquinoline-1-oxide (4-NQO) induced chromosome damage and apoptosis in bone marrow cells of Swiss albino mice. Two (EAF; 80 and 160 mg/ kg body weight - bw) or three (HF and CF; 80, 160 and 320 mg/ kg bw) doses of solvent fractions or vehicle control (25% DMSO in water) were administered orally for five consecutive days. Upon the sixth day, 4-NQO was injected intraperitoneally. The animals were killed the following day. Other control groups were comprised of animals treated with either the vehicle control or the various doses of solvent fractions, but with no 4-NQO treatment. 4-NQO induced micro-nucleated polychromatic erythrocytes (MnPCEs) in all the test groups. However, pre-treatment of animals with the solvent fractions significantly reduced the 4-NQO-induced MnPCEs as well as the percentage of apoptotic cells. The reduction of both MnPCE and apoptosis was more evident following the pre-treatment of animals with 160 mg/kg bw EAF.

In the present study, the protective effects of various solvent fractions of Mentha spicata on 4-NQO induced chromosome damage and apoptosis in mouse bone-marrow cells were investigated.

Animals
Swiss albino mice of either sex, 10 to 12 weeks old and 25-30 grams were used. All animals were obtained from the King Institute, Chennai, India, and were kept in the Institute's animal house under standard environmental conditions (temperature: 22 ± 2°C and 12 h light/ dark period). Their maintenance was in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. The Institute's ethical committee approved all the experiments.

Chemicals
4-Nitroquinoline N-oxide (4-NQO), Giemsa stain, May-Grunwald stain and Annexin V-FITC assay kit were purchased from Sigma-Aldrich, USA. All the other solvents used in the experiments were of analytical grade.

Procedure for solvent fractionation
M. spicata L. was commercially purchased and identified at the Center for Advanced Studies in Botany, University of Madras (voucher number-855). The different solvent fractions (SF) such as the hexane fraction (HF), chloroform fraction (CF) and ethyl acetate fraction (EAF) were fractionated from the ethanol extract of dried-leaf powder of M. spicata by the Villasenor et al. (2003) method. The procedure was briefly given in our previous paper (Arumugam et al., 2008a).

Experimental design for the micronucleus test
The test doses of solvent fractions such as HF, CF and EAF were determined on the basis of their estimated LD 50 doses and on preliminary laboratory experiments. These doses corresponded to 50, 25 and 12.5% of the LD 50 ( Table 1).
The femurs from each animal were dissected and the proximal heads removed. The contents of both femurs were flushed out and pooled into a total volume of 3 mL pre-filtered foetal-calf serum. The cell suspensions were sedimented by centrifugation, the supernatant discarded and the cells themselves resuspended in a small volume of fresh foetal calf serum. A droplet from the suspension was transferred to a glass microscope slide and a smear prepared. At least four smears were prepared from each animal and scored according to standard May-Grunwald staining (Schmid, 1975).

Experimental design for detection of apoptotic cells
The maximum effective doses of solvent fractions against 4-NQO induced MnPCEs were used. The experiments consisted of four groups, each group being comprised of six mice of either sex (Table 2).
Both femurs were removed and the bone marrow cells flushed out with 2 mL of phosphate buffered saline (PBS). These cells were then washed twice with PBS by gentle aspiration. The pellet was obtained following centrifugation 848 Arumugam and Ramesh  for 5 min at 2000 rpm (4°C), and was then re-suspended with 500 mL of fresh PBS. According to annexin V-FITC assay kit procedure, 1 x 10 6 cells were placed into one mL of binding buffer, to which 5 mL of annexin V -FITC were added, followed by 10 mL of propidium iodide (PI-20 mg per mL). After mixing the cell suspension, the cells were incubated in the dark for 15 min at room temperature. They were immediately analyzed by flow cytometry (Darzynkiewicz et al., 2001). Four different groups of cells were measured (Figure 1). The total number of apoptotic cells was calculated by the addition of both early and late apoptotic ones.

Statistical analysis
Results were presented as the mean ± standard error for six mice of each group. Statistical analyses were performed by one-way ANOVA using SPSS Software Version 12.0. The Student-Neuman-Keuls test (SNK TEST) was applied to assess for differences among the groups. Values of p £ 0.05 were considered to be significant.

Results
The protective effects of solvent fractions of M. spicata against 4-NQO induced micro-nucleated polychromatic erythrocytes (MnPCEs) of mouse bone-marrow are presented in Table 3. The genotoxin 4-NQO enhanced MnPCE frequency by~4 times the control value, 15.78 MnPCEs/2500 PCEs. Treatment with solvent fractions alone was ineffectual against MnPCE frequency (p > 0.05). Pre-treatment with the hexane fraction significantly reduced the mutation rate from about 58 to 42%. Dose differences were significant only at higher doses. The chloroform fraction also significantly reduced 4-NQO induced mutation frequency. The reduction was greatest at 320 mg per kg bw (about 61%) and lowest at 80 mg per kg bw (about 45%). Nevertheless, the ethyl acetate fraction showed the highest reduction of MnPCEs induced by 4-NQO at half the dose, i.e. 160 mg per kg bw when compared to the other two fractions. Even the lowest dose, 80 mg per kg bw was also effective in reducing MnPCE frequency (about 51%) which was comparable to the CF and higher than the HF at their dose of 160 mg per kg bw.
Moreover, the study of flow cytometry gave support to the proposition that 4-NQO induced chromosome dam-Protective effects of M. spicata on 4-NQO induced chromosome damage 849 age in bone-marrow cells. Four groups of cells were estimated by using the annexin V-FITC assay kit (Figure 1). The total of apoptotic cells, including those of both early and late apoptosis, was arrived at after 24 h of treatment with 4-NQO ( Figure 2). The total of apoptotic cells was found to be 12% superior to that of the control (2.65%). Treatment with solvent fractions alone did not reveal any effect on bone-marrow apoptosis. However, pretreatment with solvent fractions showed statistically significant decreases in apoptotic cells induced by 4-NQO. The highest reduction was also found to be in the EAF (3.24%) followed by CF (6.87%) and HF (7.51%). Overall, the results indicated that EAF was more effective against 4-NQO induced chromosome damage than the other two fractions.

Discussion
The protective effect of solvent fractions of M. spicata was evaluated against 4-nitroquinoline-1-oxide (4-NQO) induced chromosome damage in mice bone marrow. 4-NQO is a potent mutagen that exerts its genotoxicity in two ways. In the first and during metabolism, 4-NQO is converted into 4-hydroxyaminoquinoline 1-oxide (Ac-4-HAQO) and interacts with DNA at N2 and C8 of guanosine and also at N6 of adenine forming adducts (Han et al., 2007). As a consequence, the helical structure of DNA changes, resulting in micronuclei/chromosomal breakage (Diekmann et al., 2004). The second pathway occurs through oxidative stress caused by the formation of reactive oxygen species (ROS), such as superoxide and hydroxyl radicals, whereat 4-NQO undergoes redox cycling that brings about modified bases and DNA strand breaks (Zhang et al., 2008). Hence, 4-NQO enhanced the frequency of MnPCEs by about four times that of the control group (15.78 MnPCEs/2500 PCEs; Table 3). The observed PCE/NCE ratio is presented in Table 4. The 4-NQO group is the only one with a significant reduction in PCE/NCE ratio compared to the control. Pre-treatment with solvent fractions effectively decreased 4-NQO enhanced MnPCE frequency by about 42%-65%, depending on the dose and solvent fraction tested. Moreover, the reduction of MnPCE by solvent fractions corresponded well with their PCE/NCE ratio. The maximum reduction of 58% was observed for HF and 61% for CF at the dose of 320 mg per kg bw. Thus, there were large amounts of pigments and less phenol in the two fractions (Arumugam et al., 2008b). Pigments (carotenoids and xanthophylls) are known to exert antimutagenic and anticarcinogenic activities either by trapping 4-NQO intermediates or by the inhibition/degradation of its metabolizing enzymes. Ferraz et al., (2005) observed that lower polar fractions, such as hexane and chloroform, exerted more effective anti-proliferation due to pigments than higher polar fractions (ethyl acetate).
Among the solvent fractions, the highest reduction of MnPCEs (about 65%), was observed for EAF at a dose of 160 mg per kg bw. Even at a lower dose (80 mg per kg bw), EAF possesses a more pronounced antigenotoxic effect when compared to the highest doses of CF and HF. In contrast, CF from the ethanol extract of M. cordifolia was shown to exercise the highest MnPCE reduction against tetracycline-induced genotoxicity followed by EAF and HF. The efficacy of CF was reported to be in the presence of b-sitosterol (Villasenor et al., 2003). Hence, we found that as to the protective effect of solvent fractions against 4-NQO induced chromosome damage, the order was EAF > CF > HF, which was well in accordance with their corresponding order of antioxidants and secondary metabolites. The effectiveness of EAF could be due to the large phenol and flavonoid content (Arumugam et al., 2008b). 850 Arumugam and Ramesh  They are all well known antioxidants involved in protecting against DNA damage induced by ROS (Kumar and Chattopadhyay, 2007). In a previous study, it was also observed that EAF displayed more intense anti-inflammatory activity than the other fractions (Arumugam et al., 2008a). Furthermore, data from the study on flow cytometry also gave support to the concept on the protective effects of solvent fractions on 4-NQO induced chromosome damage. DNA damage is known to be one of the hallmarks for the formation of apoptosis (Mazur et al., 2002). During genotoxic stress, cells undergo apoptosis in the case of DNA damage being beyond repair. 4-NQO can induce apoptosis by way of the p53-dependent mitochondrial signaling pathway (Han et al., 2007). The results clearly indicated that MnPCEs induced by 4-NQO (~4 times higher than control - Table 3) was reflected in the form of total apoptotic cells (~4 times higher than control value; Figure 2). Even, the PCE/NCE ratio was also reflected by 4-NQO induced apoptotic cells (Table 4). However, pre-treatment with EAF significantly reduced 4-NQO induced apoptosis to a greater degree than with the other two fractions.
In summary, among the three solvent fractions, EAF exerted the highest protective effect against 4-NQO induced chromosome damage in mice, since EAF was found to be a powerful antioxidant due to the high level of polyphenols (Arumugam et al., 2006), this maybe constituting the means of protecting bone-marrow cells in mice from 4-NQO toxicity. On other hand, polyphenols have been reported to inhibit 4-NQO induced free radicals (Srinivasan et al., 2007). Hence, further study is called for in order to clarify what is the mechanism of EAF against 4-NQO induced chromosome damage in mice.