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Toxicological evaluation of imidazole following direct exposure to bone marrow mesenchymal stem cells.

Introduction

Imidazole an alkaloid from plant secondary metabolites, is an important component of antifungal, anti protozoal, anti helminthic and anti cancer medications. It is also a potential candidate for imparting functionality to scaffolds in tissue engineering application as this aromatic five membered hetrocyle ring compound forms the main structure of a large number of highly significant biomolecules such as the essential amino acid histidine, Vit B-12, a component of DNA base structure (purines), histamine and biotin [1].

The bioactivity of Imidazole derivatives are attributed by the parent basic heterocycle ring. Exposure of these chemicals to any organism will eventually be transduced into biological responses at the cellular level. Invitro toxicity tests thus helps in reckoning these immediate responses and translating into predictive toxicological profile of the chemical. These assays also provides a means of discerning relative toxic potencies of the chemical exposed under controlled dosimetery conditions and are especially useful for rating a chemical.

Cell culture systems have an advantage that they allow a closer look at the basic physiology of the cells interacted with and the mechanisms could be investigated in detail [2]. Compared to conventional invivo methods that mainly assess the damage due to long term exposure of the chemical to the cells as a result of bioaccumulation, actual toxic effect of the compound following acute direct exposure could be monitored with the use of cell culture systems. Several invitro assays have been validated to demonstrate toxic endpoints of the cell such as altered metabolism, decreased growth, apoptosis, cell proliferation in addition to genotoxicity and epigenetic events [3].

Recently, Stem cells have emerged as an excellent platform for defining predictive toxicity of chemicals. Adult Stem cells, particularly mesenchymal stem cells are multipotent progenitor cells found in bone marrow and various other tissues and possess a wide differentiation potential [4]. The interaction of pure Imidazole with mesenchymal stem cell might provide new insights on the toxic potential of Imidazole in creating any stem cell related pathologies. And also for a chemical whose invivo toxicity is well established, investigations with specialized cells will prove useful in clarifying the mechanism underlying its toxic action.

Hence, in this study, an attempt is made to elucidate the toxicological response of pure Imidazole following direct contact with bone marrow mesenchymal stem cells. The toxicological parameters assessed are cytotoxic evaluation, oxidative stress response as well as genotoxic evaluation.

Materials and Methods

Isolation, culture and phenotypic characterization of mouse bone marrow stem cells (BMSCs)

Mesenchymal stem cells (MSCs) were isolated from mouse bone marrow as per accepted protocol following Institutional Animal Ethics committee approval [5]. Briefly mouse bone marrow mesenchymal stem cells were isolated from an aspirate of bone marrow harvested from the femoral marrow compartment of Swiss albino mice (6-8weeks) and then cultured in a medium with Dulbecco's modified Eagle's medium (DMEM) containing high glucose (HG) and 15% fetal bovine serum (FBS) for 3 h in a 37[degrees]C, 5% C[O.sub.2] incubator. Non adherent cells and debris were removed carefully after 24 h and the adherent cells were cultured continuously. Confluent primary cultures were trypsinized (0.25% trypsin-EDTA at 37[degrees]C for 5 min) and serially passaged. Initially cultures were morphologically heterogeneous and contained hematopoietic cells of bone marrow origin and therefore cells at passage 3 were selected for the studies. The cells were phenotypically characterized for the presence of surface marker CD90 by immunostaining and visualized by fluorescent microscopy.

Assessment of cytotoxicity of pure Imidazole using bone marrow mesenchymal stem cells

The cytotoxicity of pure Imidazole in BMSCs were assessed by morphological changes after Imidazole treatment as well as MTT (3-(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium bromide) assay. Briefly BMSCs were seeded on to 6 well tissue culture plates. Medium given was DMEM HG supplemented with 10% FBS and incubated at 37[degrees]C in a 5% C[O.sub.2] atmosphere. A semi confluent monolayer of BMSCs were treated with Imidazole at different concentrations (0.5mg/mL, 1mg/ mL, 2.5 mg/mL, 5 mg/mL, respectively) for 24 h. 1% Phenol served as positive and cells as negative control respectively. After the incubation time, cells were examined under microscope for any response. Morphology of the cells was assessed in comparison to negative and positive control. The cellular responses were scored as 0, 1, 2 and 3 corresponding to non cytotoxic, mildly, moderately and severely cytotoxic.

For quantitative assessment of cytotoxicity, MTT assay were used, BMSCs at a density of 20,000-40,000 cells/ ml were seeded on to 96 well tissue culture plates and cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% FBS. The cells were treated with different concentrations of Imidazole (0.5mg/mL, 1mg/ mL, 2.5mg/mL, 5mg/mL, respectively) for 24h. Cells supplied with culture media alone served as negative control and 10 % triton X was used as positive control. After the incubation period, the metabolically active cells were quantified using MTT assay and compared with untreated control. For MTT assay, 100 [micro]l of MTT dye (50 [micro]g/mL in PBS) was added to each well in dark and incubated for 4 h at 37[degrees]C. The assay is based on metabolic reduction of soluble MTT by mitochondrial dehydrogenase to insoluble colored formazan product. The optical density was measured spectrophotometrically after dissolution in dimethyl sulfoxide (DMSO) and recorded at 540 nm using a microplate reader

Assessment of oxidative stress response of bone marrow mesenchymal stem cells following Imidazole treatment.

Oxidative stress experienced by the cell can be assessed by the measurement of level of intracellular reactive oxygen species (ROS). The generation of reactive oxygen species was monitored by employing 2,7,dichlorodihydrofluorescein diacetate (DCF-DA) [Invitrogen] which is non fluorescent unless oxidized by the intracellular ROS. The cells were by preincubated with DCF-DA at a concentration of 100 [micro]M. followed by treatment with Imidazole at different concentrations (0.5mg/mL, 1 mg/mL, 2.5 mg/mL, 5mg/mL, respectively) for 2 h, at 37[degrees]C. Hydrogen peroxide (0.09% [H.sub.2][O.sub.2]) treated cells were used as positive control for DCF-DA analysis, Cells were then washed twice in serum-free medium and resulting fluorescence were quantitated in a fluorescence microplate reader using an excitation wavelength of 488 nm and emission wavelength of 53 nm. The values were normalized to the negative control (BMSCs alone) and respective fold change was calculated.

Assessment of genotoxicity of Imidazole using bone marrow mesenchymal stem cells by chromosomal aberration test

In vitro chromosomal aberration test was performed on treated as well as untreated bone marrow mesenchymal cells. The cells were seeded in 25 [cm.sup.2] tissue culture flasks. After 24 h of incubation, they were treated with Imidazole at different concentrations (1mg/ml, 2.5 mg/ml, 5 mg/ml respectively). The test compound was allowed to remain in the cultures for 24 h. To arrest cells in metaphase 12.5 [micro]L of 0.1 [micro]g/mL colchicine (Sigma) was added to all cultures 2 h before harvest and chromosome preparations were made as described earlier [6].The frequency of the cells with structural and numeric chromosomal aberrations were scored in 50 well spread metaphase for each dose. Types of structural chromosomal aberrations were classified into following groups: chromatid breaks (ctb) chromosome breaks (csb), chromatid and chromosomal gap (ctg) The final results were judged as follows: negative (-) if the frequency of aberrant cells was <5%, inconclusive ([+ or -]) if [greater than or equal to]5 % but <10 % and positive (+) if [greater than or equal to] 10 %.

Assessment of DNA damage in bone marrow mesenchymal stem cells following Imidazole treatment by fast halo assay and DNA laddering assay.

Assessment of DNA damage in BMSCs following Imidazole treatment was also done by fast halo assay (FHA), a modification of the alkaline halo assay technique following the procedure of Sestili et al [7]. In brief, the cell suspension was diluted with an equal volume of 1% low-melting point agarose in PBS and immediately sandwiched between an agarose coated slide and a coverslip. After gel formation on ice, the coverslips were removed and the slides were immersed in 300m M NaOH for 15 min at room temperature. Ethidium bromide (2.5mg/mL) was directly added during the last 5 min of incubation. The slides were then washed and destained for 5 min in distilled water and examined immediately under fluorescence microscope. DNA after treatment was isolated from treated as well as untreated cells using genomic DNA isolation kit (Gen Elute mammalian Genomic DNA Mini prep Kit) as per kit protocol. The isolated DNA from all the samples were quantified in biophotometer and were resolved on 1% agarose gel in 1 X TAE (Tris-acetate-EDTA) buffer at 100 V. A lane was loaded with DNA ladder for reference. DNA was stained with aqueous solution of EtBr, visualized and photographed under a UV transilluminator.

Statistical analysis

Results were expressed as mean [+ or -] SD. The Student t test was performed to compare mean values. Probability of null hypothesis less than 5% (P [less than or equal to] 0.05) was considered statistically significant.

Results and Discussion

Stem cells are specialized cell which have the ability to self-renew and have the potential to differentiate into one or more specialized lineages [8]. In the adult bone marrow (BM), in addition to pluripotent hematopoietic stem cells (P-HSCs), there is another population of cells called multipotent stromal cells which are widely recognized as mesenchymal stem cells (MSCs) [9]. Even though they are present in various adult tissues, isolation from bone marrow is considered as the "gold standard" for stem cell culture and propagation. From bone marrow stroma MSCs can be isolated, expanded in culture system, and stimulated to differentiate into, cartilage, bone, muscle, fat, tendon and a variety of other connective tissues. Owing to their plasticity and ability to differentiate to multiple cell types both in vitro and in vivo they stand as an excellent source of stem cells for various biomedical applications [9]. Recently mesenchymal stem cells are also exploited as an in vitro model system to predict the toxicity of chemicals [10].

[FIGURE 1 OMITTED]

The BMSCs were isolated from a heterogeneous population based on their peculiar property of adherence to plastic surfaces and were successfully propagated in primary culture and in serial culture on tissue culture plates. They attached in colonies and spread out from individual colonies. The isolated cells demonstrated fibroblastic morphology which when confluent appeared as spindle shaped cells (Fig. 1a). Medium given was DMEM high glucose supplemented with 15% fetal bovine serum. Phenotypic charactersiation of BMSCs were done by demonstrating the cell surface marker CD90 by immunostaining (Fig 1b).

Primary cultures are widely used because the cells retain their specialized functions better than cell lines. Some of the investigations have been undertaken to validate the use of the cell systems for screening procedures and for measuring potential toxic effects of chemical on particular tissue and for assessing the mechanism of action of these chemicals on cell morphological alterations are used as an index of toxicity [11]. BMSCs could also be exploited for its ability to change surface markers in response to external stimuli as a parameter in assessing the impact of the chemical on the cell fate. More over the multi lineage differentiation potential of these cells could to mimic a number of tissue systems thereby reduce the number of animals used for the drug screening procedures.

[FIGURE 2 OMITTED]

Microscopic observation of Imidazole treated BMSCs showed that the characteristic fibroblastic appearance of the cells were lost and the cells seem to be detached from the culture surface when exposed to higher concentration (5mg/mL). However, the morphology of Imidazole treated cells at a concentration of 0.5mg/mL, 1mg/mL and 2.5 mg/mL were comparable to that of negative control (Fig 2) MTT assay also substantiate this finding that the cell viability was less than 70% in higher dosage treated cells (Fig 3a).

The first and most readily observed effect following exposure of cells to toxicants is morphological alteration in the cell in monolayer culture. A crude index of toxicity is cell viability. A cell viability measurement in comparison with the control provides an index of lethality of the test compound. Cells were exposed with different concentrations of pure Imidazole for 24 h. The OD value of control cells (Imidazole free) was taken as 100% and then calculated as the percentage of reduction of OD in Imidazole exposed cells. Morphology of BMSCs fibroblastic-like shape were changed to a round appearance and the cells were detached from the surface in higher concentrations of pure Imidazole as observed by light microscopy.

The result obtained on ROS generation is that pure Imidazole treated cells showed a dose dependent gradual decrease in ROS production compared to positive control. Interestingly, the reactive oxygen species measured were less than the negative control (Fig 3b). Reactive oxygen species (ROS) are unstable molecules that are normally produced during cell metabolism. Contradictly, higher intracellular ROS can adversely affect the cellular functions leading to oxidative stress and cell death. In Imidazole treated cells ROS production showed a decreased fold change when compared to negative control, suggesting the antioxidant property of Imidazole and Imidazole derivatives. Based on the results, it is suggested that the mechanistic toxicity observed in higher concentrations of Imidazole treatment might be attributed by other means than oxidative stress and ROS generation DNA was extracted using genomic DNA isolation kit and was quantitated. Mitomycin C was used as positive control (PC), and chemical free media as negative control. The Imidazole treated cells had a DNA content of 3.1mg/ mL, 3.5mg/mL, 3.5mg/mL, 3.8mg/mL for 0.5, 1, 2.5, 5 mg/mL Imidazole treatment Interestingly, the amount of DNA was higher in positive control and treated samples, compared to that of negative control. Positive control treated cells had a DNA content of 4.2 mg/mL while negative control had 2.3mg/mL of DNA.DNA ladder assay showed that, after 24h treatment Imidazole treated DNA samples were presented as a smear whose length showed a dose dependent trend. Complete DNA damage was observed in positive control as anticipated by showing a clear smear formation (Fig 4). Fast Halo assay is used for the assessment of DNA damage at the single-cell level. Fig 5 represents the photographs of halo assay performed on DNA samples isolated from Imidazole treated cells. It was shown that cells treated with higher concentration of Imidazole (1mg/ml, 5mg/ml) showed a halo around cell nucleus demonstrating DNA damage in the cells due to the toxic effect exerted by pure Imidazole at high concentration. Pure Imidazole exposed BMSCs cell culture system showed a slight genotoxic effect on the cells as assessed by chromosomal aberration and DNA damage. DNA quantification revealed that the concentration of DNA is higher in positive control and treatment group compared to negative control. This may be due to Copy-number variations (CNVs) which is a form of structural variation due to alterations of the DNA of a genome that results in the cell having an abnormal number of copies of one or more sections of the DNA. However, further experimentation is needed to prove this concept.

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

DNA ladder assays showed smears for both positive control and treated cell. This point to the possibility that Imidazole is able to cause random double stranded breaks in the bone marrow mesenchymal stem cell DNA by some direct or indirect mechanism. However specific band were absent in the DNA sample of chemical treated cells in the ladder assay. This is concurrent with the previous findings that the apoptosis is preceded by acidification, which activate an acid endonuclease that has some role in chromatin digestion during apoptosis. Imidazole being a base can neutralize this pre acidification step and delays apoptosis at lower concentration [12] and this study also support the finding that Imidazole induced cell death is not associated with oligonucleosomal DNA fragmentation-a hall mark of apoptosis [13]. However to validate the effect of Imidazole on early apoptosis further study is required.

Single-cell level DNA damage could be demonstrated by fast halo assay. The damaged DNA is separated from intact one by means of solvent gradient, stained with Ethidium bromide and visualized under a fluorescent microscope. By analyzing the diameter of halo, the level of DNA breakage can be calculated. Halo around the cells due to damaged DNA were observed in Imidazole treated groups of higher concentrations (Fig 5). This damage may be double stranded or single stranded which needs to be analyzed further.

[FIGURE 5 OMITTED]

Visible changes to chromosome structure and morphology is considered as indicators of genetic damage. In the present study, the number of metaphases were low all the samples, could be attributed to the discrepancies in synchronization of cell cycle in invitro MSC culture. Total 50 metaphases were counted and frequency is reported in Table 1. A representative figure of metaphase of the samples is given in Fig 6. Chromosomal aberration test on higher doses were not performed as mitotic cells could not be retrieved from culture due to toxicity of pure Imidazole at high concentration.

[FIGURE 6 OMITTED]

Karyotyping of mouse chromosome revealed that chromosome number in mice is 40 (19 autosomes and X and Y sex chromosome). Autosomes are telocentric and Y chromosome is acrocentric. In the present study, cell cycle was not synchronized and hence the number of metaphases was low in both treated and negative control cell samples. Even though when compared to negative control, 1mg/ml treated cells showed an increased frequency of chromatid aberrations. But the results could be concluded as there were no significant change At higher concentrations of Imidazole, harvest of cells for evaluation were not possible due to massive cell death. Chromosomal aberration is principally due to double strand breakage (DSB). DSBs can lead to broken chromosomes, mutations and chromosome rearrangements. All these invitro assays were used to detect the primary toxicological effects of pure Imidazole on bone marrow mesenchymal stem cells

Conclusion

In this study we have found that BMSCs in vitro responded to the Imidazole and the results obtained agrees with the previous studies and findings related to the effect of Imidazole on different cell. The cell culture is a model of a target tissue in the body and thereby mimics the tissue response to the exposure of chemicals. Stem cells have the ability to differentiate into multiple lineages. Hence by changing the experimental conditions and stimuli, these stem cells can mimic a number of tissue systems and thereby reduce the number of animals used for the chemical screening procedures. Provided that the time and degree of exposure (concentration/ exposure time in cell tests) in the experiments correspond to human exposure, these models can also potentially predict any type of chemical interference with corresponding aspects of the human body. Hence we suggest that the in vitro bone marrow mesenchymal stem cell culture can act as a good alternative to in vitro models in range-finding and screening tests which is justifiable on scientific, ethic and economic basis.

References

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[4.] Sandra A.J., Roobrouck V.D., Verfaillie C.M. and Gool S.W.V., Immunological characteristics of human mesenchymal stem cells and multipotent adult progenitor cells, Immunology and Cell Biology, 91, 32-39 (2013).

[5.] Soleimani M. and Nadri S., A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow, Nat. Protoc. 4 (10) 102106 (2009).

[6.] Dusinska M., Kazimyrova A., Barancokova M., Beno M., Smolkova B., Horska A., Raslova K.L., Wsolova L. and Collins A.R., Nutritional supplementation with antioxidants decreases chromosomal damage in humans. Mutagenesis, 18: 371-376 (2003).

[7.] Sestili P., Martinelli C. and Stocchi V., The fast halo assay: an improved method to quantify genomic DNA strand breakage at the single-cell level. Mutat. Res. 607, 205-214.(2006).

[8.] Fuchs E., and Segre J.A., Stem cells: A new lease on life. Cell 100:143-155.(2000).

[9.] Caplan A.I., Mesenchymal stem cells and gene therapy. Clin Orthop 379(suppl):S67-S70 (2000).

[10.] Remya N.S., Syama S., Gayathri V., Varma H.K., Mohanan P.V., An in vitro study on the interaction of hydroxyapatite nanoparticles and bone marrow mesenchymal stem cells for assessing the toxicological behavior, Colloids and Surfaces B: Biointerfaces, 117 389-397 (2014).

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[12.] Furlong I.J., Ascaso R., Rivas A.L. and Collins M.K.L., Intracellular acidication induces apoptosis by stimulating ICE-like protease activity, Journal of Cell Science, 110, 653-661 (1997).

[13.] Takekawa F., Nagumo T., Shintani S., Hashimoto K., Kikuchi H., Katayama T., Ishihara M., Amano O., Kawase M. and Sakagami H., Tumorspecific Cytotoxic Activity and Type of Cell Death Induced by 4-TrifluoromethylImidazoles in Human Oral Squamous Cell Carcinoma Cell Lines, Anticancer Research 27: 4065-4070 (2007).

T.R. Reshmitha, N.S. Remya, P.V. Mohanan *

Toxicology Division, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram 695 012, Kerala, India

* Corresponding Author: Dr. PV. Mohanan; Email: mohanpv@sctimst.ac.in

Received 26 June 2014; Accepted 27 June 2014; Available online 1 July 2014
Table 1: Frequency of chromosomal aberrations in Imidazole
treated cells

                           No. of
                         metaphase    Chromatid    Chromatid
Sample                    counted        gaps        breaks

Neg.Control                  50           1            0
Imidazole at 0.5mg/mL        50           0            0
Imidazole at 1mg/mL          50           6            8
Imidazole at 2.5mg/mL           Could not be evaluated
Imidazole at 5mg/mL             Could not be evaluated

                         Chromosome    Chromosome
Sample                      gaps         breaks      Fragments

Neg.Control                   0             0            0
Imidazole at 0.5mg/mL         0             0            0
Imidazole at 1mg/mL           0             1            2
Imidazole at 2.5mg/mL            Could not be evaluated
Imidazole at 5mg/mL              Could not be evaluated
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Title Annotation:Original Article
Author:Reshmitha, T.R.; Remya, N.S.; Mohanan, P.V.
Publication:Trends in Biomaterials and Artificial Organs
Article Type:Report
Date:Jul 1, 2014
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