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Studies on the Genotoxic Effect of Nickel Chloride in Mice and the PossibleProtective Role of Soybean Seeds Extracts 1
Maha A. Fahmy, 2Nagwa H.A. Hassan, 3Farouk R. Melek, 3 Zeinab M. Hassan and 3Hanan A. Al-Ashaal
Department of Genetics and Cytology, National Research Centre, Dokki, Cairo, Egypt 2 Faculty of Science Ain Shams University, Cairo, Egypt 3 Department of Chemistry of Natural Compounds, National Research Centre, Dokki, Cairo, Egypt
Abstract: Nickel is an essential trace element. Nickel and its compounds have many industrial applications. Concern about nickel compounds is due to potential occupational and environmental health hazards. In the present study the mutagenic potential of nickel chloride (NiCl 2) was tested to evaluate somatic and heritable disorders which may extend to the next generations and associated with increased risk of cancer. The protective role of dietary soybean seeds and its extracts was also evaluated. The study examined the induction of chromosomal aberrations in bone marrow cells and mouse spermatocytes and the effect on morphological sperm abnormalities. The results showed that NiCl2 induced an increase in the percentage of chromosomal aberrations in somatic and germ cells with dose and time relationship. A significant (P<0.01) increase in the percentage of abnormal sperm was observed after treatment with the tested doses. The results also demonstrated that soybean seeds and its extracts have a protective role and reduced the mutagenic potential induced by NiCl 2. Methanol extract and methanol fraction of soybean seeds gave the most effective protection. Key words: Nickel Chloride Mutagenicity Role Soybean Seeds Extracts
High quantity of nickel has been reported to show various toxicities such as pulmonary, renal and cardiovascular effects [2-4]. Carcinogenic and mutagenic potential of nickel were demonstrated . Nickel can cross the placental barrier, affecting directly the developing embryo or fetus in experimental animals . It affects various aspects of reproduction  and has immunosuppressive effect . Some nickel compounds have been established as human carcinogens based on epidemiological evidence, which show high incidence of nasal and lung cancers in refinery workers  and its potency to induce tumors in a variety of mammalian species . The carcinogenicity of Ni compounds is believed to be associated with their solubility and cellular uptake. Water insoluble Ni compounds tend to be carcinogenic than Ni soluble compounds because they are phagocytize by cells resulting in intracellular delivery of high quantities of Ni . However, the carcinogenicity of a compound is
Nickel is a natural element of the earth’s crust; therefore small amounts are found in food, water, soil and air. Individuals also may be exposed to nickel in occupations involved in its production, processing and use, or through contact with every item such as nickel containing jewelry and stainless steel cooking and eating utensils and by smoking tobacco. Smoking 40 cigarettes daily, for example, may result in inhalation of 2-23µ g of nickel. Nickel is found in ambient air at very low levels as a result of releases from oil and coal combustion, west and sewage sludge incineration, nickel metal refining, manufacturing facilities and other sources . Nickel (Ni+2) in the body is an essential trace element required in micro quantity. Nickel plays some important role in biological system such as enzyme activity, hormonal control and also in DNA, RNA and protein structure and function .
Corresponding Author: Maha A. Fahmy, Department of Genetics and Cytology, National Research Centre, Dokki, Cairo, Egypt .
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closely related to its genotoxic and mutagenic potential. In mammalian cells, the induction of sister chromatid exchanges and chromosomal aberrations, DNA-single strand breaks and DNA- protein cross-links were demonstrated with various nickel salts [12-15]. Nowadays, the beneficial diets are of particular importance since they may have a role in the prevention against many diseases and cancer. Soybean (Glycine max) is one of the major agronomic crops in many countries. Soybean containing up to 40% protein can be considered as a protein concentrate even without defate. In comparison with other plant proteins, soybean protein is lysine-rich and is therefore useful as a supplement to cereals and also as low cost quality protein for human meals. Soybean is highlighted now as a source of pharmaceutical raw materials and drugs. Various soybean contents are proved to have immune-improving, anticancer, antimutagenic effects and are potent against a broad spectrum of diseases [16, 17]. The present study concerning with, evaluating the genotoxic effects of NiCl2 in somatic and germ cells of male mice, detecting the possible protective role of soybean seeds extracts and correlating this activity with the chemical composition of these extracts. The study assumes importance in view of widespread human exposure to nickel compounds.
It is noteworthy that the average food intake of each mouse (25g) is 4.5±0.5g/day. In groups of experimental animals which feed soybean seeds, the seeds were mixed as 30% with the solid diet of animals under test. So,1.5g of soybean seeds are required/mouse/day. This amount of food intake from soybean was taken into consideration as bases for calculating the doses of soybean seeds extracts in all experiments. Soybean extracts was prepared as follows: Soybean seeds (2Kg) were covered with methanol and left for three days at room temperature. The process of extraction was repeated 3 times. The solvent from combined methanol extract was evaporated under reduced pressure. The residue obtained (159.9g) was dissolved in dist water at the desired concentration just before use (Methanol Extract- M.E. Another batch of soybean seeds (2kg) was extracted with n-hexane to give 266.6g of n-hexane fraction (n-hx.F.) after evaporation and dryness and used in cytogenetic experiments. For preparation of chloroform fraction (Ch.F.) of soybean, the plant material after extraction with n-hexane was left to dry and then extracted with chloroform (3 times). The residue obtained after dryness (23.5g) was used in cytogenetic study after dissolving in corn oil at the desired concentration just before use. The plant material, after extraction first with n-hexane, then with chloroform was left to dry. The dried material was then extracted with methanol (3 times) to give the methanol fraction of soybean seeds(M.F.).The obtained residue was weighted (2Kg gave 173.3g ) and dissolved in dist water at the desired concentration just before use. Isolation and Identification of active constituents were performed,described and published in Al-Ashaal et al. 
MATERIAL AND METHODS Animals: Male white Swiss mice ( Mus Musculus ), aged 9-12 weeks were used in all experiments.The ethical approval for the experiments was obtained from the ethical committee of the National Research centre, Cairo, Egypt. The animals were obtained from a closed random bred colony at the National Research Centre. The mice used for any one experiment were selected from mice of similar age (± 1 week) and weight (± 2g). Animals were housed in polycarbonate boxes with steel-wire tops (not more than five animals/ cage) and bedded with wood shavings. Ambient temperature was controlled at 22± 3°C with a relative humidity of 50 ± 15% and a 12- h light/dark photoperiod. Food and water were provided ad libitum.
Treatment and Cytological Preparations: Chromosomal Aberration: Single-dose Treatment: Mice received a single i.p treatment with NiCl2 at dose levels 2.625, 5.250, 10.50 and 21.00 mg Kg 1b,wt (equivalent to 1/16, 1/8, 1/4 and ½ of the experimental LD50 ).
Chemicals: Nickel chloride (NiCl2) anhydrous was purchased from Merck-Schuchardt Co. Germany.
Repeated -Dose Treatment: Mice received a daily i.p treatment with one of the doses 2.625, 5.250 and 10.50 mg Kg 1 bwt NiCl2 for 1,2 and 3 weeks and feed normal diet. With respect to the dose 5.250 mg Kg 1bwt NiCl2 (1/8LD50), in addition to NiCl2-treated groups, also the effect of (NiCl2 + M.E), (NiCl2 +n-hx.F.), (NiCl2 +Ch.F.) and (NiCl2 + M.F.) were tested. All extracts were given orally. Another groups of mice received the same dose of NiCl 2 but feed diet mixed with 30% soybean seeds.
Solvents for Extraction: n-hexane, chloroform, methanol. Plant Material and Extract Preparation: soybean seeds obtained from the Agriculture Research Centre (Egypt) were used. The seeds were autoclaved at 107°C for 15 min, let to cool then it were ground in an electric grinder. 626
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Slide Preparation and Scoring: Mice were i.p injected with colchicine 2-3h before sacrificied. Bone-marrow preparations were made according to the technique described by Yosida and Amano . A group of five mice was used for each treatment and 75 well spread metaphases were analyzed/animal for scoring different types of abnormalities. Chromosomal preparations from testes were made according to the technique developed by Evans et al . 100 well-spread diakinesis metaphase I cells were analyzed per animal to assess abnormalities in five mice/group. Scoring was performed under (25×100) X magnification with a light microscope (Litz, Germany).
RESULTS Effect of Nickel Chloride: Chromosomal Aberration in Bone Marrow: The results in Tables (1,2) show that different doses induced significant percentage of chromosomal aberrations. Such percentage was found to be dose-and time dependent. The maximum percentage reached 28.26± 0.32 (P<0.01) 3 weeks after treatment with the dose 10.50 mgkg 1bwt NiCl2 compared with 4.5±0.32 for control. Different types of chromosome aberrations were recorded. Chromosomal Abnormalities in Spermatocytes: Nickel chloride at the tested doses induced a statistically significant percentage of chromosomal abnormalities in mouse spermatocytes (except 24h after single i.p treatment with the dose 2.625). This percentage increased with increasing the dose and with longer duration of treatment. The maximum percentage of abnormalities reached 17.00 ± 0.71 (P< 0.01) which represents about 4.8 fold increases compared with the negative control 3.60 ± 0.51. Nickel chloride affected separation in univalents, X-Y univalent dominated. Fragments/ breaks, gaps and Translocations as chain IV were observed (Tables 3, 4 )
Sperm -Shape Abnormalities: Groups of five mice each were i.p. treated with NiCl2 daily for five consecutive days at dose levels of 2.625, 5.250 and 10.50 mg Kg 1 b.wt and feed normal diet. Other groups of mice treated with the same doses but feed normal diet mixed with 30% soybean seeds. Animals were sacrificed 35 days after the first treatment by cervical dislocation. Sperm were prepared according to the recommended method of Wyrobek and Bruce . The epididymides were excised and minced in 2ml physiological saline, dispersed and filtered to remove large tissue fragments. Smears were prepared and stained with 1% Eosin Y. A total of 1000 sperm were counted /animal, scoring different types of sperm abnormalities. Scoring was performed under magnification (25×40) X with light microscope. In all Experiments negative (non-treated) and positive (Endoxan) control groups were run alongside the test material. Control groups treated orally with different soybean seeds extracts were also tested.
Sperm -Shape Abnormalities: The results show that the three tested doses of NiCl2 induced a statistically significant increase in the percentage of sperm abnormalities (P < 0.01) which show positive correlation with the dose Table (5). Various morphological sperm abnormalities in heads and tails were observed. Some heads may acquire unusual shape; others show a reduced or increased size. Coiled tail abnormality was also recorded.
Statistical Analysis: The significance of the difference between experimental and control data was calculated using t-test. The resulting data between groups (NiCl2treated and NiCl2+soybean) were compared by ANOVA, followed by Duncan’s multiple-range test.
Protective Role of Soybean: Figures 1 and 2 show the percentage of chromosomal aberrations in mouse bone-marrow and spermatocytes, comparing the
Table 1:Chromosomal aberrations induced in mouse bone marrow cells 24h after a single i.p. treatment with different doses of nickel chloride (NiCl2). Abnormal metaphases
Number of metaphases with different types of aberrations
375 metaphases examined in five mice per treatment.
R.T= Robertsonian translocation;
M.A. = metaphases with more than one type of aberration. ** Highly significant P < 0.01 level
and or break
Global J. Pharmacol., 8 (4): 625-634, 2014 Table 2: Percentage of metaphases with chromosomal aberrations induced in mouse bone-marrow cells after repeated i.p. treatment for 1,2 and 3 weeks with different doses of nickel chloride (NiCl2) and fed normal diet. Abnormal metaphases -------------------------------------------------------------Treatment and doses I- Control (non-treated)
Mean% ± S.E.
Mean% ± S.E.
4.5 ± 0.32
1.82 ± 0.32
13.86 ± 0.54**
2.93 ± 0.78
19.73 ± 0.54**
6.64 ± 0.60**
22.10 ± 0.90**
7.70 ± 0.78**
19.73 ± 0.51**
7.70 ± 0.79**
21.86 ± 0.98**
8.80 ± 0.99**
23.73 ± 0.51**
10.40 ± 0.50**
23.20 ± 0.60**
10.90 ± 0.63**
24.50 ± 0.99**
14.13 ± 0.90**
28.26 ± 0.65**
14.66 ± 0.85**
II- Nickel chloride 1 week treatment: 2.625 5.250 mg kg
10.50 2 weeks treatment: 2.625 5.250 mg kg
10.50 3 weeks treatment: 2.625 5.250 mg kg
** Significant at (0.01) level (t-test). Table 3: Chromosomal abnormalities induced in mouse spermatocytes 24h after a single i.p. treatment with different doses of Nickel chloride (NiCl2) Abnormal Metaphases
Number of metaphases with different types of aberrations
500 metaphases examined in five mice per treatment.
M.A. = metaphases with more than one type of aberration. ** = Highly significant P < 0.01 level.
Table 4: Percentage of metaphases with chromosomal abnormalities induced in mouse spermatocytes after repeated i.p. treatment for 1, 2 and 3 weeks with different doses of nickel chloride (NiCl2) and fed normal diet. Treatment and doses
I- Control (non-treated)
Examined metaphases No. 500
Abnormal metaphases No. 181
Abnormal metaphasesMean% ± S.E. 3.60 ± 0.51
6.20 ± 0.37**
9.60 ± 0.75**
11.20 ± 0.73**
9.00 ± 0.84**
13.20 ± 0.49**
14.00 ± 0.55**
9.40 ± 0.51**
15.20 ± 0.73**
17.00 ± 0.71**
II- Nickel chloride 1 week treatment 2.625 5.250 mg kg
10.50 2 weeks treatment: 2.625 5.250 mg kg
10.50 3 weeks treatment: 2.625 5.250 mg kg
10.50 ** Significant at (0.01) level (t-test).
Global J. Pharmacol., 8 (4): 625-634, 2014 Table 5: Morphological sperm abnormalities induced in male mouse after i.p. treatment with different doses of nickel chloride (NiCl2). Tail
Abnormal sperms -----------------------
Head abnormalities -------------------------------------------------------------------------------------------
abnormali - ties
Mean ± S.E
I. control (non treated )
2.46 ± 0.14
4.28 ± 0.31** 5.12 ± 0.23**
6.38 ± 0.20**
2.22 ± 0.36
2.32 ± 0.22 2.42 ± 0.24
3.20 ± 0.48
3.14 ± 0.13*
III. Mice fed
(+ ve control)
No of different types of sperm abnormalities -----------------------------------------------------------------------------------------
5000 sperm were counted five mice per treatment (1000 sperm per mice)
* Significant P < 0.05 level
** Highly significant P < 0.01 level.
Fig. 1: Percentage of chromosomal abnormalities (including gaps) induced in mouse bone-marrow cells after i.p. treatment with NiCl2 at 5.25 mg kg 1 bw , NiCl2+soybean seeds feeding and NiCl2 with "M.E.", "n-hx. F.", "Ch.F." or "M.F." of soybean seeds for 1 week (A), 2 weeks (B) and 3 weeks (C)
Fig. 2: Percentage of chromosomal abnormalities induced in mouse spermatocytes after i.p. treatment with NiCl2 at 5.25 mg kg 1 bw , NiCl2+soybean seeds feeding and NiCl2 with "M.E.", "n-hx. F.", "Ch.F." or "M.F." of soybean seeds for 1 week (A), 2 weeks (B) and 3 weeks (C). It is worth mentioning that, controls orally administered soybean extracts have the same results as control non-treated after cytological examination results obtained in mice treated with NiCl2 at the dose 5.25 mg Kg 1b.wt (1/8 LD50) and feed normal diet and that treated with NiCl2 and feed diet mixed with soybean seeds or orally treated with different soybean extracts (Methanol extract, n-hexane fraction, chloroform fraction or methanol fraction).
The results show that feeding mice with soybean seeds reduced the percentage of chromosomal aberrations in NiCl 2-treated groups in bone marrow and mouse spermatocytes. The results also revealed variable effects of soybean extracts and fractions. All of them reduced the percentage of 629
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NiCl2-induced chromosomal aberrations. Methanol extract and methanol fraction gave the most effective protection. With respect to sperm abnormalities, Table( 5) shows that feeding NiCl -treated mice with soybean seeds
chromosomes. The absence of bivalents and the formation of unpaired chromosomes at the time of induction of the first anaphase, may lead to random segregation of homologous chromosomes i.e aneuploidy . The results show that X-Y univalent was more frequent. This type of abnormality has been discussed as an indicator of male sterility . Translocations in the form of Chain IV were observed. The maximum percentage reached 5% after repeated treatment with the highest tested dose which is relatively high. Translocations are one of the commonest forms of rearrangement in man. Fragments/breaks and gaps were also recorded in a low percentage. Morphological sperm abnormalities with dosedependent manner were observed in the present study. Both head and tail abnormalities were recorded. The head abnormalities most probably reflect a change in DNA content . Coiling of sperm tail mainly involves its orientation, which give an impression of a reduced sperm movement. Such limitation in sperm movement was reported to reduce fertility in both animals  and humans . It was reported that non-motile sperms lacked the dynein arms which is an ultra-structural abnormality of the microtubules in the tail motor complex . There is evidence that coiled-tail abnormality induced by other metals .The results of the present work are coincide well with the finding of other authors who demonstrated dose-dependent changes in sperm motility, count and abnormalities in mice treated with nickel chloride [33,34]. Oxidative stress mechanisms are speculated to play a significant role in nickel-induced genotoxicity . Nickel compounds induce genetic instability and chromosome instability which could result in multiple genetic alterations and are critically involved in carcinogenesis . Nickel II is also able to significantly enhance the genotoxicity of other mutagens and carcinogens  and interfere with DNA-repair mechanism and this may contribute to its genotoxicity . The results also demonstrated a significant decrease in the percentage of chromosomal aberrations and sperm abnormalities in all treated-mice feed soybean or orally administrated soybean extracts. Our results are in agreement with the previously reported data concerning the antimutagenic properties of soybean. Soybean reduced chromosomal damage and aberrations, DNAbreaks, point mutation and the effect on mitotic indices induced in mammalian cells by chemical mutagens e.g. the carbamate insecticide sevein ; the nitrosamine
(during 35 days until sacrificed) was effective in reducing different kinds of sperm abnormalities induced by NiCl 2. DISCUSSION The results of the present study showed that nickel chloride at the tested concentrations induced a significant increase in the percentage of chromosomal abnormalities in bone marrow and mouse spermatocytes after single and repeated i.p treatments, with a dose and time- response. The repeated dosing caused a higher percentage of chromosomal aberrations than the single-dose treatment which indicates a cumulative effect of NiCl2. Similar results have been reported after repeated treatments with other metals, e.g. lead acetate, potassium dichromate, cobalt chloride and beryllium chloride . Different types of chromosomal aberrations were observed in bone marrow cells after treatment with NiCl 2. Such results are in agreement with the previously published data demonstrated various types of aberrations included gaps, breaks and exchanges in FMA mouse mammary carcinoma cells, in cultured Chinese hamster ovary(CHO) cells, in mouse bone marrow and cultured spleen cells.The resulting data also confirm the findings of other authors who reported that inhalation exposure of Wister rats to metal aerosol derived from nickel refinery waste for a period of 4- months resulted in a significant increase in chromosomal aberrations in alveolar macrophages of Wister rats indicating its genotoxicity. Genetic effect of NiCl2 in somatic cells was also demonstrated by increase in the frequency of sister chromatid exchanges (SCE?s) and induction of micronuclei in peripheral blood and mouse bone marrow [14, 26]. Epidemiological studies also showed mutagenic risk in workers exposed to nickel compounds as evidenced by increasing in the frequency of chromosomal aberrations and SCE's and inhibition of DNA-repair synthesis . Nickel chloride induced chromosomal abnormalities in mouse spermatocytes which give information on transmissible genetic damage. It was observed that dissociated univalents dominated (autosomal and X-Y univalents). The process of chromosome pairing and the formation of bivalents during meiosis are necessary for proper genetic recombination and segregation of 630
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precursors, dibutylamine and sodium nitrate  and acetoxyacetylaminoflurene . Fahmy et al  reported that, feeding mice with soybean diet reduced the genotoxicity of potassium dichromate as indicated by a significant reduction in the induced SCE's, chromosomal aberrations in bone marrow and spermatocytes and sperm abnormalities. Studies on the protective role of soybean showed that the anticarcinogenic/antimutagenic properties of soybean depend on its high nutritional value and to the presence of some active ingredients. Soybean is an excellent source of protein and amino acids. The protective role of amino acids for minimizing the incidence of chemically induced tumors was reported  Soybean is also a good source of ascorbic acid, nicotinic acid, vitamin A, thiamin, riboflavin and contain fibers .Vitamin C is especially important because it combats the effect of many chemical toxins including heavy metals and participating in DNA-repair processes . Soybean fibers (non starch polysaccharide) are regarded to have the property of eliminating mutagens by absorbing them and thus have no chance to reach the target cells . Protease inhibitors are another natural ingredients demonstrated in soybean. It was reported that the Bowman-Birk (BBI) which derived and isolated from soybean is the most potent one of the known protease inhibitors. It can contract the formation of oxygen radicals and play a major role in the prevention and or treatment of several diseases in addition to cancer, at very low concentrations [17, 44]. Phytic acid content also seems to be responsible for some of the antimutagenic properties of soybean. It is a highly charged antioxidant and has antitumor effects . Soybean contains non-nutrient bioactive phytochemicals that have health promising and disease preventing properties like isoflavones, saponins, coumestans and lignans. Concerning the protective effect of various soybean extracts, the results revealed that the methanol fraction and methanol extract gave the most effective protection and maintained all parameters studied at near the control values. Preliminary phytochemical studies on the prepared methanol fraction and extract revealed the presence of isoflavones glycosides and saponins as the main constituents. Anthony  reported that 90% of isoflavones could be extracted by alcohol. Isoflavones have been proposed to be the active component responsible for the beneficial effects of soybean foods and appear to work in conjunction with certain peptides or protein fractions from soybean to protect against many diseases and cancer . Isoflavones of soybean
possessed 4 to 5 folds antioxidant activity as ascorbic acid. Such antioxidant properties can be mediated by scavenging and/or suppressing the formation of reactive oxygen and/or nitrogen species and up- regulating antioxidant defense mechanism . The principle isoflavones in soybean are genistein and daidzein and their metabolities. These constituents seem to hold the most promise from the therapeutic stand point. Genistein and daidzein derived from soybean have antimutagenic/anticarcinogenic properties [50,51] and might exert their effect via hormonal regulation activity, controlling cell enzymes and inducing apoptosis [51,52]. Furthermore, genistein inhibits angiogenesis, the growth of blood vessels needed for tumors to enlarge . Saponins, the other major constituent detected in the prepared methanol fraction/extract were reported to have antimutagenic activity against mutagens induced DNAdamage and exhibit more potent activities than vitamin E and C [54, 55]. Soyasaponins appear to have anticancer properties by virtue of their antioxidant and antimutagenic properties . CONCLUSION In view of the results of the present work and the literature reviewed concerning the genotoxicity of nickel chloride, it can be concluded that the environmental contamination by this industrial metal may bring serious problems to human health including genetic hazards. The results also demonstrated the potential role of soybean and its components as natural protective antimutagenic agents and open the door for developing new drugs from this natural source. REFERENCES 1. 2. 3.
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