Introduction

Nordihydroguaiaretic acid (NDGA) is a phenolic lignan present in the evergreen shrubs Larrea divaricata and Guaiacum officinale (Fig. 1) [1]. NDGA possesses a number of interesting biological properties which are of potential use to humans such as enzyme inhibition [2], antimicrobial properties [3], protection from neurotoxicity and bladder toxicity [4, 5], stimulation of the corpus luteum for the secretion of progesterone [6], potential vaso- and bronchodilation [7], and anti-apoptotic [8], estrogenic [9], antimutagenic [10] and anti-cancer [11] properties. NDGA has also been reported to possess both genotoxic [12] and anti-genotoxic potential [13] in vivo and in vitro. Chlormadinone acetate (CMA), a synthetic progestin, has been reported to induce genotoxic damage in both in vivo [14] and in vitro studies [15]. In the present study, the effect of NDGA was studied on the frequencies of chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs) induced by CMA in mouse bone-marrow cells.

Materials and methods

Chemicals

NDGA (CAS no.: 500-38-9, Fluka), CMA (CAS no.: 302-22-7, Sigma), Hoechst 33258 stain (0.05% w/v), N-methyl-N′-nitro-N-nitroso-guanidine (1.2 × 104 μg/kg body weight) and 5-bromo-2-deoxyuridine (1.6 g/kg body weight) were purchased from Sigma. Giemsa solution (5 and 7%) in phosphate buffer (pH 6.8) and DMSO (0.1 ml/animal) were purchased from E. Merck, India.

Animals

Swiss albino female mice (Mus musculus L.), 25–30 g and 10–12 weeks old, were procured from Lucknow (UP), India, and grouped in different polypropylene cages (five animals per group) at a mean temperature of 25°C. Permission was granted for experimentation by the departmental ethical committee.

Analysis of sister chromatid exchanges

The fluorescent plus Giemsa technique was followed for SCEs analysis [16]. 5-Bromo-2-deoxyuridine (1.6 g/kg body weight) in tablet form was implanted subcutaneously in the neck region of each mouse under mild anaesthesia; 30 min later, CMA at 22.50 mg/kg body weight, and NDGA at 1, 5 and 10 mg/kg body weight were separately injected intraperitoneally (i.p.). DMSO and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) were used as negative and positive controls, respectively. After 21 h, the animals received an i.p. injection of colchicine (6.0 mg/kg body weight), and 3 h later, the bone marrow of both femurs was collected and stored in KCl 0.075 M at 37°C for 30 min. The supernatant was removed by centrifugation, and 5 ml of fixative (methanol:glacial acetic acid, 3:1) was added. The fixative was removed and the procedure was repeated twice. The microscope slides were prepared by air drying and were stained for 20 min in a 0.05% (w/v) Hoechst 33258 solution, rinsed with tap water and placed under a UV lamp for 90 min, and then covered with Sorensen’s buffer, pH 6.8, and stained with 5% Giemsa solution in phosphate buffer (pH 6.8) for 15 min. The SCE average was obtained from the analysis of metaphases in the second cycle of divisions. At least 60 second-division metaphases per mouse were scored to determine the frequency of SCEs. The slides were scored blindly by one scorer.

Chromosomal aberrations analysis

For the analysis of CAs, the tested and control doses were the same as described for the SCE analysis. Twenty-one hours after the treatment, the animals were injected i.p. with colchicine (6.0 mg/kg body weight), 2 h before killing. Bone-marrow preparations for the analysis of CAs in metaphase cells were obtained by the technique of Yosida and Amano [17]. The slides were stained with 7% Giemsa stain in phosphate buffer (pH 6.8). Five animals were included in each treatment group, and 100 well-spread metaphases were analysed per animal for CAs. The slides were scored blindly by one scorer.

Statistical analysis

Statistical analysis was performed by using one-way analyses of variance (ANOVA).

Results

In the SCEs analysis, a clear dose-dependent decrease in the number of SCEs per cell was observed when CMA (22.50 mg/kg body weight) was given along with different doses of NDGA (1, 5 and 10 mg/kg body weight) (Table 1). NDGA itself did not significantly increase the number of SCEs per cell at 1 mg/kg body weight but at 5 and 10 mg/kg body weight, SCEs per cell were slightly significantly different.

Fig. 1
figure 1

Structure of nordihydroguaiaretic acid

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Table 1 Frequency of SCEs in mice bone-marrow cells
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In CAs analysis, a dose-dependent significant decrease in the number of abnormal cells was observed when CMA (22.50 mg/kg body weight) was treated separately with three doses of NDGA (1, 5 and 10 mg/kg body weight) (Table 2). No significant increase in the number of abnormal cells was observed at 1 mg/kg body weight of NDGA but at 5 and 10 mg/kg body weight of NDGA the numbers of abnormal cells were slightly significantly different (Table 2).

Table 2 Mean percentage of chromosomal aberrations in mice bone-marrow cells
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Discussion

The results of the present study reveal that NDGA reduced the genotoxic damage by CMA in mouse bone-marrow cells. CMA, a synthetic progestin, is used either as a single entity drug or in combination with estrogens, such as ethinyl estradiol or mestranol in oral contraceptives [18]. Prolonged use of oral contraceptives has been reported to induce different types of cancer in both animals and humans [19–22]. The use of synthetic progestins cannot be completely eliminated, but their genotoxic effects can be reduced by the use of antioxidants [23, 24] and natural plant products [25–27]. CMA induces genotoxic effects by the generation of reactive oxygen species (ROS) in human lymphocytes in vitro [15]. In our earlier study, three doses of CMA, 5.62, 11.25 and 22.50 mg/kg body weight, were studied and the doses at 11.25 and 22.50 mg/kg of body weight were found to be genotoxic. The LD50 for CMA obtained was 90 mg/kg [14].

NDGA has an antitumor capacity which is probably related to the inhibition of lipoxygenase activity. It also inhibits cytochrome P450-dependent monoxygenases which metabolise pre-carcinogens to carcinogens [12]. The antigenotoxic activity of NDGA seems to depend on the specific mutagen involved and on its biological characteristics. Several explanations can be given for its antigenotoxic activity; one of them relates to its participation as enzyme inhibitor in the arachidonic cascade as well as in the P450-dependent monooxygenases. This inhibition may eliminate the bioactivation of promutagens acting in this way. A second property of NDGA is its phenolic structure, which relates to its antioxidant action, as well as its capacity for trapping free radicals [13].

NDGA possesses antioxidant [28] and free radical-scavenging properties [7, 29]. CMA generates hydroxyl radicals by nucleophilic reaction [15], and NDGA is a potent scavenger of hydroxyl radicals, superoxide anions and singlet oxygen [29]; as a result, NDGA reduced the genotoxic damage of CMA in mice. The selected doses of NDGA in the present study were based on the study performed by Madrigal-Bujaidar et al. [12]. They reported the LD50 for NDGA as 282.2 mg/kg. The selected doses in the present study (1, 5 and 10 mg/kg body weight) were much lower than one-fourth of the LD50 value.

Plants are a good source of medicines and are associated with the modulation of mutagenic potential of various substances [30]. Genotoxicity testing is useful in providing human risk assessments. Many of the CAs observed in cells are lethal, but there are many corresponding aberrations that are viable and cause either somatic or inherited genetic effects [31], which lead to cancer or other chronic degenerative processes. CMA produces mammary tumors in dogs [32] and increases the incidence of mammary gland hyperplasia and mammary nodules [33]. It forms DNA adducts in rat and human hepatocytes in vitro [34–36] and also induces micronuclei in rat liver cells in vivo [20]. Antioxidants are known to reduce mutagenic potential of various mutagens in in vivo studies [37, 38]. The identification and characterization of various active principles can help frame important strategies to reduce the risk of cancer in human beings [39]. NDGA, a phenolic lignan, is potent enough to reduce the genotoxic effect of CMA in mice bone-marrow cells, but it should be used within a careful dose range so that the desired pharmacological effects can be achieved without any toxicity.