Introduction

Boron, the fifth element in the periodic table, is a naturally occurring element widely distributed in the earth’s crust. It is found in the environment primarily combined with oxygen to form compounds called borates. They are commonly found in nature, and are present in oceans, sedimentary rocks, coal, shale, and some soils (Argust 1998; Turkez et al. 2012a). Boron compounds are used in borosilicate glass products, but are also used in agriculture, in fire retardants, and in soaps and detergents (Richold 1998). In recent years, there is a growing interest in the biological activities of boron compounds. New studies demonstrated that boron compounds strengthen the antioxidant defense mechanism (Pawa and Ali 2006; Geyikoglu and Turkez 2008; Celikezen et al. 2014). In addition, it has been reported that boron compounds (such as borax, boric acid) are non-genotoxic agents on human blood cells (Turkez et al. 2007, 2010; Geyikoglu and Turkez 2008; Turkez 2008). Furthermore, boron compounds have attracted much interest with respect to their protective effect against free radical damage that may be the cause of many diseases including cancer (Korkmaz et al. 2007; Mahabir et al. 2008).

The exploring of novel natural or synthetic antioxidants becomes a very popular research area in the World since the last 20 years (Geyikoglu et al. 2005; Geyikoglu and Turkez 2005); in addition, many efforts have been performed to explore new antioxidant-featured compounds (Cingolani et al. 2000; Cacciatore et al. 2003, 2005; Rispoli et al. 2004; Turkez and Sisman 2007; Heuking et al. 2009; Di Stefano et al. 2009a, b; Sozio et al. 2010; Turkez et al. 2012b). Newly, scientists and researchers are interested in the medical application of boron compounds. But the data on their physiological effects and risk potentials in animals and human is very limited (Geyikoglu and Turkez 2007; Colak et al. 2011). On the other hand, lithium metaborate dihydrate (LMD) is used as nonlinear optic material, in cell phones and pagers (Schubert and Brotherton 2006). As much as we know, there is no study about cytogenetic and oxidative potentials of LMD. Therefore, the aim of the present study was to firstly evaluate the cytogenetic (MN and CA assays) and oxidative effects (TAC and TOS analysis) of LMD on human peripheral blood cultures for its possible and safe use in several industries including chemical, glass, electronic, cosmetic and pharmaceutic industry.

Materials and methods

Production of lithium metaborate dihydrate

Lithium metaborate dihydrate (LMD) was produced according to the following equation;

$$ {\text{Li}}\left( {\text{OH}} \right){\text{aq}} + {\text{H}}_{3} {\text{BO}}_{3} \left( {\text{aq}} \right) \to {\text{LiBO}}_{2} \cdot 2{\text{H}}_{2} {\text{O}} $$

The produced LMD was analyzed by using the titrimetric method to determine the B2O3 content of the sample (Snell and Hilton 1968). It was found that the produced LMD content of B2O3 was about 40.56 % (Fig. 1).

Fig. 1
figure 1

Lithium meta borate dehydrate TG/DTA decay curve

Full size image

Experimental design

Whole heparinized human blood from five healthy non-smoking male donors between the ages of 24 and 27 with no history of exposure to any genotoxic agent was used in our experiments. Questionnaires were obtained for each blood donor to evaluate exposure history, and in addition, each donor signed informed consent forms. For all volunteers involved in this study, hematological and biochemical parameters were analyzed and no pathology was detected (Evans and O’Riordan 1975). 10 ml of peripheral blood was collected from each donor aseptically in a sodium-heparinized syringe. We added 6 ml of PB-MAX Karyotyping Medium (Gibco, Carlsbad, CA, USA) to each culture tube to be used for the assay. Then we added 0.5 ml heparinized blood to each tube. Various concentrations (0, 1.25, 2.5, 5, 10, 20, 40, 80, 160, 320, 640 and 1,280 mg/l) of LMD were tested in blood cultures. The concentrations were selected according to previous studies (Turkez et al. 2007; Arslan et al. 2008; Kahraman et al. 2013). MN and CA rates were assessed in peripheral lymphocytes and the method that was used for the preparation of the peripheral lymphocytes is explained in sections below. Experiments were conformed according to the guidelines of the World Medical Assembly (Declaration of Helsinki). The cultures without LMD were studied as control− group. Mitomycin C (10 μM, Sigma-Aldrich, Steinheim, Germany; Vijayalaxmi et al. 1996) was used as the positive control in MN and CA assays. Ascorbic acid (10 μM, Sigma, St. Louis, MO, USA; Turkez 2011) and hydrogen peroxide (25 μM, Sigma; Benhusein et al. 2010) were also used as positive controls in total antioxidant capacity (TAC) and total oxidant status (TOS) analysis, respectively.

TAC and TOS analysis

The major advantage of this test is to measure the antioxidant capacity of all antioxidants in a biological sample and not just in the antioxidant capacity of a single compound (Kusano and Ferrari 2008). Since the measurement of different oxidant molecules separately is not practical and their oxidant effects are additive, the TOS of a sample is measured and this is named total peroxide, serum oxidation activity, with reactive oxygen metabolites or with some other synonyms. The automated Trolox equivalent TAC and TOS assays were carried out in plasma samples obtained from blood cultures for 24 h by commercially available kits (Rel Assay Diagnostics, Gaziantep, Turkey).

Micronucleus (MN) assay

Human lymphocytes were stimulated by LMD and cultured in a 37 °C incubator with a humidified atmosphere of 5 % CO2 for about 72 h. After 44 h LMD stimulation, cytochalasin B (Sigma, MO, USA; final concentration of 6 mg/ml) was added. Whole blood cells were harvested by centrifugation, treated with a hypotonic solution [0.075 M KCl (Merck, Darmstadt, Germany) at 37.4 °C]. Then the culture tubes were centrifuged at 2000 rpm for 5 min, the supernatant was discarded, and the pellet was resuspended using 10 mL of fresh fixative solution (methanol and acetic acid, 3:1 (Merck, Darmstadt, Germany)). The tubes were centrifuged at 2000 rpm for 5 min and the supernatant was discarded. This procedure was repeated 3 times. The resulting cells were re-suspended and dropped onto clean slides. To prepare the slides, 3–5 drops of the fixed cell suspension were dropped on a clean slide and air-dried. The slides were stained with Giemsa (Sigma, St Louis, MO, USA) in phosphate buffer (pH 6.8) and scored. MN was scored in 1,000 bi-nucleated cells and the frequency of cells with micronuclei was determined (Fenech and Morley 1985).

Chromosomal aberration (CA) assay

CA tests were performed not only to study the cytotoxicity of the material on cells but also to determine the aberrations induced by the particular material on chromosomes of the human lymphocytes cell line. Human lymphocytes were stimulated by LMD and cultured for about 72 h in a 37 °C incubator with a humidified atmosphere of 5 % CO2. Two hours prior to harvesting, 0.1 ml of colchicine (0.2 mg/ml, Sigma, St Louis, MO, USA) was added to the culture flask. Cells were harvested by centrifugation, treated with a hypotonic solution [0.075 M KCl (Merck, Darmstadt, Germany), at 37.4 °C]. Again, the culture tubes were centrifuged at 2000 rpm for 5 min, the supernatant was discarded, and the pellet was resuspended using 10 mL of fresh fixative solution (methanol and acetic acid, 3:1 (Merck, Darmstadt, Germany)). The tubes were centrifuged at 2000 rpm for 5 min and the supernatant was discarded. This procedure was repeated 3 times. The resulting cells were re-suspended and dropped onto clean slides. To prepare the slides, 3–5 drops of the fixed cell suspension were dropped on a clean slide and air-dried. The slides were stained with Giemsa (Sigma, MO, USA) in phosphate buffer (pH 6.8). For each treatment, 30 well-spread metaphases were analyzed to detect the presence of chromosomal aberrations. Criteria to classify the different types of aberrations (chromatid or chromosome gap and chromatid or chromosome break) were in accordance with the recommendation of EHC (Environmental Health Criteria) 46 for environmental monitoring of human populations (IPCS 1985).

Statistical analysis

Statistical analysis was performed using SPSS software (version 16.0, SPSS, Chicago, IL, USA). The Duncan’s test was used to determine whether any treatment significantly differed from controls or each other. Statistical decisions were made with a significance level of 0.05.

Results

The serum TAC and the TOS were evaluated by using an automated colorimetric measurement method. As shown from the results presented in Table 1, four concentrations of LMD (5, 10, 20 and 40 mg/l) resulted a significant increase of TAC levels in cultured human blood cells compared with the controls. On the other hand, LMD did not change the TOS levels in cultured lymphocytes at all the concentrations.

Table 1 The level of total antioxidant capacity (TAC) and total oxidant status (TOS) in human blood cultures treated with LMD for 24 h
Full size table

Results obtained from the analysis of MNs and CAs in human lymphocytes were cultured with no treatment (Control−), were treated with MMC (Control+) or with different LMD concentrations are shown in Figs. 2 and 3, respectively. LMD at the tested concentrations did not induce significant (P > 0.05) number of MNs and CAs frequencies. However, the MMC applied culture (as positive control) showed about three fold increases of both parameters as compared to the control− group.

Fig. 2
figure 2

Micronucleus (MN) rates in human lymphocytes after treatment with LMD in vitro. (Positive control: Mitomycin C (10−7 M). Values are expressed as mean ± SD for five cultures in each group. The bars marked by different letters are significantly different from each other at a level of 5 %.)

Full size image
Fig. 3
figure 3

Chromosome aberration (CA) frequencies in human lymphocytes after treatment with LMD in vitro. Abbreviations are as in Fig. 2

Full size image

Discussion

The alterations in enzyme activities and DNA damage are extensively used as significant biomarkers to assess the genotoxicity and oxidative stress of natural or man-made chemical materials (Turkez et al. 2005; Turkez and Togar 2010; Sisman and Turkez 2010; Dong et al. 2012). In the present study, for assessing the antioxidant/oxidant effects of LMD, TAC and TOS assays were performed for the first time. In fact, total antioxidant capacity is one of the suitable biochemical parameter to evaluate antioxidant status of body fluids. And the capacity in plasma is related to production or intake of antioxidants. Hence, it monitors the action potential to increased or normal status of reactive oxygen species (Miller et al. 1993; Lantos et al. 1997; Ghiselli et al. 2000; Balogh et al. 2001). This study reported that LMD treatments at low concentrations (5, 10, 20 and 40 µg/ml) caused significant increase of TAC levels, while TOS levels were not affected. To our knowledge, there is no report in literature about the antioxidant/oxidant effect of LMD on cultured human lymphocytes cells or any other cell lines. Therefore, its antioxidant/oxidant effect could not be compared with other boron compounds. According to the literature, Hunt and Idso (1999) reported that boron prevents oxidative damage by increasing of glutathione and its analogs or by supporting other neutralizing agents. Pawa and Ali (2006) administrated borax to rats as a boron source followed by administration of thioacetamide. As a result they reported that boron might have positive effects on the oxidant/antioxidant balance. Boron was shown to exhibit ameliorative effects against cyclophosphamide induced lipid peroxidation and genotoxicity by enhancing antioxidant defense mechanisms in rats (Ince et al. 2014). Besides, Ince et al. (2010) found that boron supplementation decreases lipid peroxidation and enhances antioxidant defense mechanism in vivo. Again, administration of boron in a dose-dependent manner reversed malathion-induced oxidative stress, lipid peroxidation and antioxidant enzyme activity in rats (Coban et al. 2014). Likewise, Turkez et al. (2012a) explored that different borates, including borax, colemanite, boric acid and ulexite, exhibited antioxidant features by using TAC assay in vitro. Similar to the present results, a very recent study indicated that potassium tetraborate showed antioxidant properties in vitro (Celikezen et al. 2014). In addition, our results revealed that 1.25 and 2.5 mg/l of LMD did not exhibit antioxidant features. Because, the observed slight increases of TAC values, were not statistically different from the control group at these two concentrations. The reason could be that the lower doses (<5 g/ml) used are close to the physiological limits. In fact, the normal value of BA in the blood has been reported as 3 mg/l (EVM, 2002).

In this study, the genotoxic potential of LMD was investigated by using the MN and CA assays. This study established that LMD was non-genotoxic because results did not indicate any significant increase in the ratios of the CAs and MNs in lymphocytes exposed to LMD as compared to control values. These findings are in agreement with previous assessments. Landolph (1985) assessed mutagenic effect of crude borax in V79 Chinese hamster cells, C3H10T ½ mouse embryo fibroblasts and diploid foreskin fibroblasts of human. At the end of the study, borax did not show genotoxic effect. Similarly, boric acid did not affect DNA synthesis in male F344 rat hepatocytes (Bakke 1991) and it was assessed for mutagenic potential in the L5178Y tk± mouse lymphoma cell and result of the study was negative (Mc Gregor et al. 1988). In addition, Türkez and Geyikoglu (2006) studied the mutagenic properties of some boronated compounds in cultured human lymphocytes. They reported that the used boronated compounds did not change the sister-chromatid exchange (SCE) frequencies or MN formations. Again, Turkez et al. (2007) evaluated the genotoxic potential of boric acid, borax, colemanite and ulexite by SCE, MN and CA assays in human blood cell cultures and found that no tested boron compounds led to genotoxicity.

As a conclusion, the present study clearly indicated for the first time that LMD has antioxidant ability with its non-genotoxic nature. However, further studies are needed to understand biological activity of LMD in different in vivo and in vitro cell models.