The application of an in vitro micronucleus test in mouse fibroblast L929 cells

The application of an in vitro micronucleus test in mouse fibroblast L929 cells

Mutat Res Gen Tox En 841 (2019) 36–42 Contents lists available at ScienceDirect Mutat Res Gen Tox En journal homepage: www.elsevier.com/locate/gento...

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Mutat Res Gen Tox En 841 (2019) 36–42

Contents lists available at ScienceDirect

Mutat Res Gen Tox En journal homepage: www.elsevier.com/locate/gentox

The application of an in vitro micronucleus test in mouse fibroblast L929 cells Ewa Drozda, Irena Bubkoa, Karolina Jaworskab, Beata M. Gruber-Bzuraa, a b

T



Department of Biochemistry and Biopharmaceuticals, National Medicines Institute, Chełmska 30/34 Str., 00-725, Warsaw, Poland Department of Applied Microbiology, Faculty of Biology, Institute of Microbiology, University of Warsaw, Miecznikowa 1 Str., Poland

A R T I C LE I N FO

A B S T R A C T

Keywords: Mouse fibroblasts L929 Micronuclei assay Cytokinesis block Cytochalasin B Phenobarbital/5,6-benzoflavone

The MNa (in vitro the micronucleus assay) is recommended for studying genotoxicity of chemicals. However, no protocol is currently available for experiments with mouse fibroblast L929 cells. The aim of this study was to improve the scope of CBMNb (cytokinesis-block micronucleus) test. Optimization consisted of: selection of a noncytotoxic concentration of cytokinesis blocker - cytoBc (cytochalasin B) and type and definition of the positive controls, verification of the efficacy of phenobarbital/5,6-benzoflavone as an S9 enzyme inducer as well as the identification of an optimal staining method. The compounds were tested in three exposure regimens: 6 h exposure with S9 activation followed by a 24 h recovery period, 6 h exposure followed by a 24 h recovery without metabolic activation of S9 and 30 h continuous exposure without S9. Different parameters, such as internal and interlaboratory reproducibility were investigated and criteria for test correctness were proposed. Higher MN rates were achieved using 1 μg/mL cytoBc as a cytokinesis blocker, and MMSd (methyl methanesulfonate), (250 μM), Cole (colchicine), (0.5 μM) and CPf (cyclophosphamide), (30 μM) as positive controls. In regard to the recommended S9 inducer, phenobarbital/5,6-benzoflavone was more effective as Aroclor 1254. Giemsa and acridine orange stains were optimal for the evaluation of MN formation. The protocol described in this study with L929 cells produced the reliable results and is suitable for performing the CBMNb experiments according to the current OECD Guideline #487.

1. Introduction Recently the OECD published Guideline #487 [1] for MN assaysa, with or without cytokinesis block. CBMNb experiments are recommended for routine tests of genotoxicity of newly developed chemicals. MN scoring has to be performed with cells that have gone through mitosis during or after treatment. Cytochalasin B is widely used to block cytokinesis because it inhibits actin assembly and thus prevents the separation of daughter cells after mitosis, leading to the formation of binucleate cells [2]. According to the guideline, currently available data underline the validity of the CBMNb test using various cell types [3–8]. However, no test protocol is currently available for the fibroblast L929 line (NCTC clone 929, ATCC® CCL-1™). This line originates from mouse C3H/An connective tissue and was one of the first lines which was established in continuous culture [9]. It is widely used in many experimental aspects, including toxicology [10–13], and is also

recommended by PN-EN ISO 10993-5 norm for the cytotoxicity assessment of biomedical devices and materials which are supposed to come in contact with human subject [14]. The aim in our laboratory was to develop a validated method for CBMNb test with L929 cells. Issues which are crucial for this procedure are: blocking of cytokinesis to stop mitosis at the stage of binuclear cells, the choice of adequate positive controls which give a reproducible and expected increase of MN, metabolic activation of promutagens and their conversion into potentially genotoxic derivatives, which is related with the activation of enzyme activation mix (S9) prepared from the livers of rodents (usually rats), and cell staining for selective visualization and counting of MN. The OECD Guideline #487 [1] recommends the use of cytoBc in the range between 3 and 6 μg/mL. However, it is stated that the appropriate concentration of cytoBc has to be determined for each cell type to achieve the optimal frequency of binucleated cells in cultures and to produce a sufficient number of cells for scoring. The optimal

Abbreviations: a MN assay, in vitro micronucleus assay; bCBMN, cytokinesis-block micronucleus; ccytoB, cytochalasin B; dMMS, methyl methanesulfonate; eCol, colchicine; fCP, cyclophosphamide; gB[a]P, benz[a]pyrene; hPCBs, polychlorinated biphenyls; iDT, doubling time; jCBPI, cytokinesis-block proliferation index ⁎ Corresponding author. E-mail addresses: [email protected] (E. Drozd), [email protected] (I. Bubko), [email protected] (K. Jaworska), [email protected] (B.M. Gruber-Bzura). https://doi.org/10.1016/j.mrgentox.2019.05.005 Received 6 November 2018; Received in revised form 16 April 2019; Accepted 10 May 2019 Available online 11 May 2019 1383-5718/ © 2019 Elsevier B.V. All rights reserved.

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concentration of cytoBc has not been determined for experiments with L929 cells. Positive controls in CBMNb tests should be used at concentrations that cause reproducible and detectable increases of the MN rates in order to demonstrate the sensitivity of the test system. In this study, several compounds were tested, namely: MMSd (an alkylating agent, clastogen), Cole (a microtubule polymerization inhibitor, aneugen), recommended for the assay without metabolic activation, (-) S9, CPf (an alkylating agent), B[a]Pg (an intercalating agent, clastogen) which were recommended for experiments with metabolic activation, (+) S9. The time-schedule of the experiments were adjusted to the doubling time (DTi). The most commonly used system for the metabolic activation of promutagens and their conversion into potentially genotoxic derivatives, is the use of a co-factor-supplemented liver enzyme mix (S9). For its preparation animals are treated with enzyme-inducing agents such as PCBh - Aroclor 1254 [8,15,16] or a combination of phenobarbital and β-naphtoflavone [17–19]. The aim of this study was to optimize the treatment conditions for CBMNb test with L929 cells. The testing methodology and assay criteria in the paper are in agreement with OECD Test Guideline #487 [1], and also with the respective PN-EN ISO 10993-3 norm [20].

(unpublished data). 2.2.2. CytoBc concentration The optimal concentration for the blocking of cytokinesis cytoBc was chosen on the basis of the results. The efficacy of cytoBc was determined by calculation of the Cytokinesis-Block Proliferation Index (CBPIj) which is the ratio between mono-, bi- and multinucleated cells and the total number of cells [1]. The cytoBc concentration is adequate when the CBPIj value is > 1.5 (> 50% of cells in the culture are binucleated). The concentration range of cytoBc tested in this part of the study was 1–10 μg/mL. 2.2.3. Positive control concentrations The optimal positive control concentrations were defined in experiments with and without S9, using S9 induced by Aroclor 1254. The acceptance criteria were defined as the concentration of the proposed substance that evokes a significant induction of MN compared to the control values. The increase of MN rates in cells exposed to the positive control is one of the key parameters of the test system. MMSd, Cole, CPf and B[a]Pg were evaluated in regard to their cytotoxic properties. Short-term or long-term treatment, (-) S9 or (+) S9 were recommended for each of the substances in OECD Guideline #487. CPf and B[a]Pg were tested with S9 mix, which was prepared as follows [3]: glucose-6-phosphate (180 mg/mL), NADP (25 mg/mL), KCl (0.15 M) and S9 were mixed in a ratio 1:1:1:2 (v/v). Aliquots of this mixture were added to the cell cultures to achieve a final concentration of 2%. The effect of two S9 preparations were compared, namely Aroclor 1254 induced and phenobarbital/5,6-benzoflavone induced. MMSd and Cole were tested (–) S9.

2. Materials and methods 2.1. Materials CytoBc from Drechsleradematioidea was purchased from MP Biomedicals; MMSd (CAS no. 66-27-3) – from Merck, CPf (CAS no. 5018-0) – from MP Biomedicals; Cole (CAS no. 64-86-8) – from SigmaAldrich; B[a]Pg (CAS no. 50-32-8) - from Merck. DMSO was from LabScan, Poland. Minimum Essential Eagle’s Medium, fetal bovine serum and 100x Antibiotic Antimycotic were obtained from Sigma, Saint Louis, USA. Trypsin-EDTA (0.5%), without phenol red was purchased from Gibco, Carlsbad, Germany. Aroclor 1254 induced male Sprague Dawley rat liver S9, in 0.15 M KCl and phenobarbital/5,6benzoflavone induced male Sprague Dawley rat liver S9, in 0.15 M KCl were from Molecular Toxicology Inc., USA. CytoBc, MMSd and CPf were dissolved and diluted in culture medium, medium with cytoBc (1 μg/mL) and medium with 2% S9, respectively. B[a]Pg was dissolved in DMSO and diluted in medium with 2% S9. The final DMSO concentration in B[a]Pg solutions did not exceed 5% (v/v) of the final culture volume (untreated controls were used to ensure that the percentage of organic solvent had no adverse effect (data not shown).

2.2.3.1. Short-term exposure. Exponentially growing L929 cell cultures were treated with the positive controls for 6 h with or without S9 mix. Following 6 h treatment, the cultures were washed with culture medium, then 1.0 mL of culture medium containing 1 μg/mL cytoBc, was added. Subsequently, the cells were incubated for 24 h before harvest. 2.2.3.2. Long-term-exposure. Exponentially growing L929 cell cultures (-) S9 were treated with positive controls continuously in presence of cytoBc (1 μg/mL) for 30 h and then harvested. 2.2.4. Metabolic activation The efficiency of the activation of the promutagens was evaluated in experiments with S9 induced by Aroclor 1254 or phenobarbital/5,6benzoflavone. An increase in MN was studied in L929 cells which were exposed to CPf (30 μM), as described in subsection 2.2.2 CytoBc concentration. All experiments were performed in quadruplicate.

2.2. Methods 2.2.1. Cell line and cultivation conditions NCTC clone 929 (L929) was acquired from the American Type Culture Collection, Manassas, VA, USA. The cells were cultured at 37°C in 5% CO2 in Minimum Essential Eagle’s Medium supplemented with 10% heat-inactivated fetal bovine serum and 1x Antibiotic Antimycotic, with a final concentration of 0.100 units penicillin, 0.1 mg streptomycin and 0.25 mg amphotericin B per mL. Cells were passaged using 75 cm2 cell culture flasks (Nunc) at a density of 2 × 106 cells per flask and maintained until reaching 90% confluence. Cell dissociation was routinely achieved in presence of Trypsin-EDTA (0.5%), without phenol red. Trypsinized cells were resuspended in culture medium and seeded into a new flask. 24 h prior to the treatment the cells were seeded into 4-well Millicell® EZ slides (Merck Millipore) at a density of 1 × 105 cells/mL in a total volume of 1 mL per well and incubated at 37°C in 5% CO2. The average doubling time (DTi) was defined as ca. 27 h ± 2 h, according to ATCC [21]. The cell line was routinely tested for Mycoplasma sp., Acholeplasma sp. and Ureaplasma sp. infections using a validated in-house qPCR assay

2.2.5. Staining The cells were harvested directly on Millicell® EZ slides for 20 min in cold methanol. After air-drying, the slides were stained with: (i) Giemsa solution (0.05% in phosphate buffer pH 6.8) for 20 min and rinsed 4x with the same buffer; or (ii) with DAPI solution (10 μg/mL in methanol) for 15 min in 37°C and rinsed with distilled water; or (iii) with ethidium bromide solution (10 μg/mL in distilled water) for 15 min in 37°C and rinsed 3x with distilled water; or (iv) with acridine orange solution (100 μg/mL in PBS) for 15 min in 37°C and rinsed 5x with PBS. The slides were evaluated using a microscope (50x objective) and white light (Giemsa) or fluorescent light (DAPI, ethidium bromide, acridine orange). 2.2.6. Acceptance criteria Three parameters were defined to characterizing the usefulness of the CBMNb test with L929 cells: (i) the maximal limit for the MN score in control cultures, (ii) the CBPIjvalues and (iii) the increase of the MN 37

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results are shown in Fig. 2. MMSd concentrations > 500 μM induced pronounced cytostatic and cytotoxic effects, preventing the scoring of MN in the cell culture (data not shown). For further experiments a concentration of 250μM was defined as optimal. The genotoxicity of Cole was evaluated in long-term experiments without S9, using 1.0 μg/mL cytoBc. The tested concentration range was 0.31 μM to 10 μM and was similar to that recommended by Fowler et al. [4] for another rodent cell line (Chinese hamster lung cells). In our studies, Cole did not have a cytotoxic effect in L929 cells in the range: 0.31 μM – 10 μM (data not shown). As shown in Fig. 3, higher MN numbers as in the control cells (> 90%) were found with all tested concentrations. In subsequent studies a concentration of 0.5 μM was used. The genotoxicity of CPf was evaluated in short-term experiments, with S9. The tested concentration range (7.81 μM – 1000 μM) was wider than that recommended by Fowler et al. [4] for CHL/IU cells. As shown in Fig. 4, a dose-related response was observed in the range between 7.81 μM and 31.25 μM. The most pronounced MN induction was observed with 31.25 μM. CPf used at concentrations between 62.5 μM and 500 μM led to strong cytostatic and cytotoxic effects, preventing the scoring of MN (data not shown). At a concentration of 1000 μM, CPf caused complete destruction of the cell culture (data not shown). In further studies the concentration of 30 μM of CPf was used. The genotoxicity of B[a]Pg was evaluated with S9. The concentration range was 7.81 μM – 500 μM. The compound caused dose-dependent cytostatic effects at levels ≥ 7.81 μM, making scoring of MN less reliable (data not shown). Concentrations between 250 and 500 μM caused severe cytotoxic effect and complete destruction of the cells (data not shown).

numbers in cells which were treated with positive controls. 2.2.7. Reproducibility The reproducibility of the CBMNg test with L929 cells was evaluated in three independent experiments. Each culture was tested in 4 repetitions, a SD ≤ 5% for the MN score was defined as the limit required to prove that the method is repeatable. 2.2.8. Reproducibility in different laboratories To confirm the reproducibility of the CBMNg test, a blind-coded sample of medical material was evaluated in two different laboratories in regard to MN generation, according to OECD Guideline #487 [1]. The acceptance limit to prove the method was reproducible was defined as a not-significant difference (p ≥ 0.05) between the MNs number induced in the cells with a given sample. 2.2.9. Statistical analyses Where possible, a total of at least 2000 binucleate cells per sample were analyzed for the presence of MN in the studies described above. The results are presented as % of MN ± SD per binucleate cell population. The significance of the induction of MN as compared to controls was determined using t-Student’s test. The consistency between the results obtained by different laboratories (reproducibility) was evaluated with the t-Student’s test for unpaired samples. Statistical significance was defined as p < 0.05. 3. Results 3.1. Optimal concentration of cytoBc Long-term treatment without S9 was performed with L929 control cells using cytoBc in the concentration range between 1 and 10 μg/mL. Incubation for 30 h (ca. 1.5 cell cycle length) is consistent with OECD Guideline #487, which recommends treatment with cytoBc for 1.5–2 cell cycle lengths. In our experiments, incubation of L929 cells with cytoBc longer > 30 h resulted in formation of many quadrinucleate cells in the culture (data not shown). After 30 h incubation, the cells were harvested and stained with Giemsa. The results are presented in Fig. 1. As shown, cytoBc at concentrations between 1 and 2 μg/mL were optimal and resulted in the presence of more than 50% binucleated cells in the cultures. No cytotoxic effects were observed. CytoBc used at concentrations 3 or 4 μg/mL had a significant cytotoxic activity. The morphology of the cells was changed and apoptotic cells were frequently observed. CytoBc at concentrations between 5 and 10 μg/mL resulted in severe cytotoxicity, which caused inhibition of growth and division of the cells. Generally, a correlation between cytoBc concentration and frequency of MN was found in the range between 1 and 3 μg/mL. 1, 2 and 3 μg/mL of cytoBc resulted in 0.78%, 7% and 8% of MN, respectively. With concentrations of 4 μg/mL, scoring of MN was difficult due to the cytotoxic effect of cytoBc. For subsequent experiments a concentration of 1 μg/mL cytoBc was used, i.e. the lowest noncytotoxic concentration, which shows the expected effect of cytokinesis block (characterized by CBPIj value > 1.5). CBPI values for both short- and long-term variants of the MN assaya were included in the range: 1.51−2.07.

3.3. Metabolic activation Comparative studies on the efficacy of Aroclor 1254 or phenobarbital/5,6-benzoflavone as the inducer of S9 were performed with CPf (30 μM) and with four samples tested in our laboratory (solutions of four nanometals). The cytoBc concentration used in these tests was 1 μg/mL. The results are presented in Fig. 5. As shown, the induction of S9 with phenobarbital/5,6-benzoflavone resulted in slightly lower MN counts as compared with Aroclor 1254. However, the reported differences were not significant. The MN frequencies obtained in L929 intact cells, in the cells with CPf, and each of the tested substances were similar regardless of the type of S9 activator. This provides that phenobarbital/5,6-benzoflavone may be an S9 activator equivalent to Aroclor 1254. 3.4. Cell staining method Four different staining methods were tested in the study, i.e.: Giemsa, DAPI, ethidium bromide and acridine orange. For Giemsa staining a very good effect was obtained. Nuclei and cytoplasm were clearly visible and distinguishable. The use of acridine orange also resulted in very good staining effects. Nuclei were easily distinguishable from the cytoplasm. In both cases, MN were easy to identify. In cells stained with DAPI, nuclei and MN were detected without difficulties, however, the cytoplasm was not clearly visible. Ethidium bromide did not give satisfactory results, as both, nuclei and cell edges were poorly visible.

3.2. Positive controls The genotoxicity of MMSd was evaluated in the short-term tests without S9, using 1.0 μg/mL cytoBc. The MMSd concentration range was 7.81 μM – 1100 μM and was defined on the basis of our earlier studies, which showed the strong cytotoxicity of MMSd in a concentration > 5000 μM (data not shown). Therefore, for the current studies lower concentrations were chosen. MMSd in the range between 7.81 μM and 250 μM caused a dose-related increase of MN rates. The

3.5. Acceptance criteria The upper limit for MN score and the CBPIj acceptance value were defined on the basis of the results obtained with three batches of L929 cells tested in one year (Table 1). MN frequencies in untreated cells were determined by the method recommended by OECD Guideline #487 [1], namely with Aroclor 1254 – induced S9 and with the positive 38

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Fig. 1. Morphology of L929 cells after 30 - h exposure to various concentrations (1–10 μg/mL) of cytoB (cytochalasin B). The cells were cultured at 37°C in 5% CO2 in Minimum Essential Eagle’s Medium supplemented with 10% heat-inactivated fetal bovine serum and 1x Antibiotic Antimycotic, with final concentration of 0.100 units penicillin, 0.1 mg streptomycin and 0.25 mg amphotericin B per mL. To visualize nuclei and MN (micronuclei), the cells were stained with Giemsa.

controls: MMSd, CPf and Cole. The cells were stained with Giemsa. In each experiment the mean MN score in untreated L929 cell cultures was in the range of 2.08–4.84%. Similarly, on the basis of our historical data, the MN rate in the untreated L929 cells was < 5%. The CBPIj value determined in the untreated cultures was ≥ 1.5 (data not shown). On the basis of these results, above criteria were defined as: < 5% for the MN score in the control cells and for the CBPIj values as ≥ 1.5.

concerning the repeatability of the method. The SD of the MN number obtained for 4 repetitions in each of three tested L929 cell cultures was far below 5% and it was included in the range 0.45–1.70% which proves that the MN assaya with the use of L929 cells is repeatable. All tests fulfilled the acceptance criteria. The positive controls and S9 inducer were used as described in the subsection 3.5 Acceptance criteria.

3.6. Repeatability

The MN scores were obtained from the blind-coded sample of medical material in two different laboratories. All tests fulfilled the SST criteria. The positive controls were used as described in the subsection

3.7. Reproducibility

The results of the CBMNb test presented in Table 1 provided the data 39

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Fig. 4. Production of MN (micronuclei) in L929 cells after 6 – h exposure to various concentrations of CP (cyclophosphamide) (short-term treatment, (+) S9). The cells were cultured at 37°C in 5% CO2 in Minimum Essential Eagle’s Medium supplemented with 10% heat-inactivated fetal bovine serum and 1x Antibiotic Antimycotic, with final concentration of 0.100 units penicillin, 0.1 mg streptomycin and 0.25 mg amphotericin B per mL. To visualize nuclei and MN (micronuclei), the cells were stained with Giemsa. The results are presented as the mean % of MN ± SD per binucleate cells population from two independent experiments, each performed in quadruplicate. “M” indicates inhibition of cell division (> 90% of mononucleated cells). “C!” indicates severe cytotoxicity and complete destruction of cell culture. Asterisks indicate significant increase of MN rates compared with control L929 cells (t-Student’s test, ns – not significant; ***p < 0.001; ****p < 0.0001).

Fig. 2. Production of MN (micronuclei) in L929 cells after 6 - h exposure to various concentrations of MMS (methyl-methanesulfonate) (short-term treatment, (-) S9). The cells were cultured at 37°C in 5% CO2 in Minimum Essential Eagle’s Medium supplemented with 10% heat-inactivated fetal bovine serum and 1x Antibiotic Antimycotic, with final concentration of 0.100 units penicillin, 0.1 mg streptomycin and 0.25 mg amphotericin B per mL. To visualize nuclei and MN (micronuclei), the cells were stained with Giemsa. The results are presented as the mean % of MN ± SD per binucleate cells population from 3 to 6 independent experiments, each performed in quadruplicate. Asterisks indicate significant increase of MN rates compared with control cells (tStudent’s test, *p ≤ 0.05; **p < 0.005; ****p < 0.0001).

Fig. 3. Production of MN (micronuclei) in L929 cells after 30 - h exposure to various concentrations of Col (colchicine) (long-term treatment, (-) S9). The cells were cultured at 37°C in 5% CO2 in Minimum Essential Eagle’s Medium supplemented with 10% heat-inactivated fetal bovine serum and 1x Antibiotic Antimycotic, with final concentration of 0.100 units penicillin, 0.1 mg streptomycin and 0.25 mg amphotericin B per mL. To visualize nuclei and MN (micronuclei), the cells were stained with Giemsa. The results are presented as the mean % of MN ± SD per binucleate cells population from 3 to 6 independent experiments, each performed in quadruplicate. Asterisks indicate significant increase of MN rates compared with control L929 cells (t-Student’s test, ****p < 0.0001).

Fig. 5. Production of MN (micronuclei) in L929 control cells and cells after 6 – h exposure to CP (cyclophosphamide) and to four tested substances (solutions of nanometals) (short-term treatment, (+) S9). S9 was activated with Aroclor 1254 or phenobarbital/5,6-benzoflavone. The cells were cultured at 37°C in 5% CO2 in Minimum Essential Eagle’s Medium supplemented with 10% heat-inactivated fetal bovine serum and 1x Antibiotic Antimycotic, with final concentration of 0.100 units penicillin, 0.1 mg streptomycin and 0.25 mg amphotericin B per mL. To visualize nuclei and MN (micronuclei), the cells were stained with Giemsa. The results are presented as the mean % of MN ± SD per binucleate cells population from the experiment performed in quadruplicate. Asterisks indicate significant increase of MN rates compared with control L929 cells (t-Student’s test, ***p < 0.001; ****p < 0.0001).

3.5 Acceptance criteria and caused a significant increase of the MN frequencies. The consistency between the researchers/lab technicians was evaluated with the t-Student test for unpaired samples. The results are presented in Table 2. The MN scores obtained in the experiment without metabolic activation are similar. The only significant difference (p < 0.0073) in the MN rates obtained with the sample in the two laboratories was found in a short-term test with metabolic activation. In conclusion, the results confirm that the MN assaya with L929 cells is reproducible and standardized.

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Table 1 Repeatability of the Cytokinesis-Block Micronucleus (CBMN) assay. The results are measured % of MN (mean from quadruplicate repetitions ± SD) per binucleate cells population obtained in the three L929 cell cultures with or without metabolic activation. Cell culture

1

2

3

CBMN without metabolic activation, % of MN

CBMN with metabolic activation, % of MN

Short-term (6 h) treatment

Mean ± SD

Long-term (30 h) treatment

Mean ± SD

Short-term (6 h) treatment

Mean ± SD

3.14 3.92 1.51 2.00 4.53 4.78 5.43 4.43 3.80 4.80 2.76 2.26

2.64 ± 1.09

1.99 1.20 1.53 2.33 6.37 4.20 2.25 3.82 4.80 4.40 4.58 5.60

2.33 ± 0.50

1.54 3.11 3.37 3.49 4.21 4.69 5.34 3.41 5.04 2.94 4.80 3.49

2.88 ± 0.90

4.79 ± 0.45

3.40 ± 1.13

4.16 ± 1.70

4.84 ± 0.53

4.41 ± 0.81

4.07 ± 1.01

Table 2 Reproducibility of the Cytokinesis-Block Micronucleus (CBMN) assay. The results are measured % of MN (mean from quadruplicate repetitions ± SD) per binucleate cells population obtained from two different laboratories in L929 cells exposed to a blind-coded sample of medical material. Laboratory I

Short-term (6 h) treatment without metabolic activation Consistency between the results from Laboratories I and II Long-term (30 h) treatment without metabolic activation Consistency between the results from Laboratories I and II Short-term (6 h) treatment with metabolic activation Consistency between the results from Laboratories I and II

Laboratory II

Control cells MN (% ± SD)

Sample MN (% ± SD)

Control cells MN (% ± SD)

Sample MN (% ± SD)

2.08 ± 1.09

1.42 ± 0.88

3.00 ± 0.61

1.99 ± 0.98

The control cells: difference between the means % of MN was insignificant, p = 0.5332 Sample: difference between the means % of MN was insignificant, p = 0.2982 2.69 ± 0.10 1.93 ± 0.81 2.22 ± 0.51 The control cells: difference between the means % of MN was insignificant, p = 0.6401 Sample: difference between the means % of MN was insignificant, p = 0.9304 3.81 ± 0.59 4.18 ± 1.53 2.13 ± 0.39 The control cells: difference between the means % of MN was insignificant, p = 0.3342 Sample: difference between the means % of MN was significant, p = 0.0073

4. Discussion

1.89 ± 1.16

1.67 ± 1.01

similar effect was observed by Fellows and O’Donovan [23] in the same cell line after treatment for 24 h with 3 μg/mL of cytoBc. In our study, in L929 cells, cytoBc at the same concentration (3 μg/mL) caused a significant increase of MN in binucleated cells. The strong dose-dependence between cytoBc and MN formation, observed in a range of cytoBc between 1 and 10 μg/mL in L929 cells, probably results from sensitivity of this cell line to this compound. The L929 cells which were used in this study showed comparable sensitivity as L5178Y cells to B[a]Pg. According to Kirkland [24], 3 h incubation of L5178Y cells with B[a]Pg in a concentration of 6 μM with S9 produced > 55% toxicity. In the experiments with L929 cells, 6 h exposure to B[a]Pg at a similar concentration (7.81 μM) caused a clear cytostatic effect. This study indicates that an alternative metabolic inducer, i.e. a combination of phenobarbital and 5,6-benzoflavone is equally as effective as polychlorinated biphenyl Aroclor 1254, which is not recommended due to its toxicity. CPf with phenobarbital/5,6-benzoflavone - induced S9 induced a MN rate number in L929 cells comparable to the seen with Aroclor 1254 - activated S9. The observed differences were not significant and did not affect the final results of the test. The results are consistent with the findings of other authors who conducted comparable studies on the efficacy of metabolic inducers [14, 17, 25]. Garcia Franco et al. [26], studied the mutagenic activity of 2-anthramine, 2-acetylaminofluorene, 3-methylcholanthrene and benzo [a]pyrene in Salmonella typhimurium with rat liver S9 activated either with phenobarbital or beta-naphthoflavone (TA98), and found that the greatest response of the bacteria cells was yielded after simultaneous treatment of the donor animals with both compounds. Further experiments are required with additional model mutagens to confirm the assumption that the use of phenobarbital/5,6-benzoflavone as S9 inducers is an suitable in general.

The OECD Guideline #487 [1] state that a cell is suitable for MN assaysa when it is demonstrated to reliably and accurately detect substances with aneugenic and clastogenic activity [22]. Mouse fibroblast cells NCTC clone L929 is the next rodent cell line that can be used in MN experiments, in addition to hamster (CHO, V79, CHL/IU) and other mouse cell lines (mouse leukemia cells L5178Y). L929 cell line is already being used in cytotoxicity tests in vitro, which are within the scope of PCA (Polish Centre for Accreditation) accreditation and European Directorate for Quality of Medicines (EDQM) attestation at the National Medicines Institute, Warsaw, Poland. It is an advantage to use the same cell line in cytotoxicity tests and genotoxicity tests, as the information about the cytotoxicity of a tested substance can be used to define concentrations for genotoxicity experiments. On the basis of this study, a protocol was developed which fulfilled the OECD criteria. Control cultures tested had very low MN frequencies (4.84% ± 0.53%). On the basis of DTi defined for L929 cells, a recovery time of 24 h after treatment with positive controls or samples is sufficient to ensure that the cells divide. In this study, exposure of L929 cells to the positive controls produced significant increases of the MN frequencies, i.e. from 20% to ≥ 95%. The high sensitivity of L929 cells to aneugen Cole is notable. In this study, 30 h treatment of L929 cells with this compound (concentration 0.31 μM), generated > 90% of MN. L929 cells seem to be more sensitive to Cole as regards MN generation as compared with CHL/IU cells. In the study of Fowler et. al. [4], a clear effect was obtained with higher doses, namely > 1.25 μM (> 0.5 μg/ mL). Oliver et al. [5] found that in presence of cytoBc the induction of MN in L5178Y cells was more obvious than in mononucleated cells. A 41

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The optimization of the staining procedure in the MN assaya is a very important issue [27]. It is known that the results of MN assaysa depend strongly on the staining methods [28]. In our studies the best effects were achieved with Giemsa and acridine orange. Both dyes allowed the nuclei to be clearly distinguishable from the cytoplasm. Counting binucleated cells and MN using these dyes was reliable and user-friendly. These results provide data regarding the specific cell response to the MN procedure. According to Polard et al. [27], acridine orange is now routinely used in rodent MN assaysa. Its suitability in experiments with L929 cells was confirmed in our study. DAPI and ethidium bromide were also studied as potential stains for MN detection in L929 cells, but these stains only allowed for the identification of nuclei and MN. The cytoplasm of the cells was not well visualized, which may be a source of error in the scoring of binucleated cells.

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