TY - JOUR
AU - Elhajouji, Azeddine
AB - Abstract In order to minimise the number of positive in vitro cytogenetic results which are not confirmed in rodent carcinogenicity tests, biological systems that are p53 and DNA repair proficient should be recommended. Moreover, an appropriate cytotoxicity parameter for top dose selection should be considered. Recent International Conference on Harmonisation draft S2 and Organisation for Economic Co-operation and Development (OECD) 487 guideline accepted the in vitro micronucleus test (MNT) as a valid alternative method for in vitro chromosome aberration test within the in vitro cytogenetic test battery. Since mitosis is a prerequisite for expression of the micronuclei, it is compulsory to demonstrate that cell division occurred, and if possible, to identify the cells that completed mitosis. The OECD guideline recommends the use of a cytokinesis block for the assessment of proliferation in primary T-lymphocytes. The work presented in this manuscript was initiated to develop a novel flow cytometry-based primary human lymphocyte MNT method. This new assay is based on a three-step staining procedure: carboxyfluorescein succinimidyl ester as a proliferation marker, ethidium monoazide for chromatin of necrotic and late apoptotic cells discrimination and 4,6-diaminodino-2-phenylindole as a DNA marker. The proof of principle of the method was performed using genotoxic and non-genotoxic compounds: methyl methanesulfonate, mitomycin C, vinblastine sulphate, cyclophosphamide, sodium chloride and dexamethasone. It has been shown that the new flow cytometry-based primary human lymphocyte MNT method is at least equally reliable method as the standard Cytochalasin B MNT. However, further validation of the assay using a wide selection of compounds with a variety of mechanisms of action is required, before it can be used for regulatory purposes. Moreover, a miniaturisation of the technology may provide an additional advantage for early drug development. Introduction In vitro cytogenetic tests may, in some experimental conditions, demonstrate high rate of irrelevant positive results in comparison to rodent carcinogenicity tests which may lead to extensive in vivo genotoxicity testing with an increased number of test articles (1). The best hope for reduction of false-positive results is using biological systems that are p53 and DNA repair proficient. In addition, an appropriate cytotoxicity parameter for top dose selection should be considered (1). Since primary human lymphocytes (HuLy) fulfil these expectations, they may be less prone than standard rodent cell lines to produce irrelevant positive results (1). Recent International Conference on Harmonisation (ICH) 2 guideline draft revision (2009) and the latest Organisation for Economic Co-operation and Development (OECD) guideline (2) accepted the in vitro micronucleus test (MNT) as a valid alternative method for in vitro chromosome aberration test (3). The MNT is a well-established assay allowing detection of aneugenic and clastogenic agents. There have been a number of attempts to automate HuLy MNT by means of image analysis (4–8) and flow cytometry (9–16). The advantages of both systems are a decreased analysis time and elimination of subjective judgement due to scorer skills. In order to improve the sensitivity and specificity of the flow cytometry-based HuLy MNT, various staining procedures have been applied (9–11,14,16–18). The flow cytometry technology is based on a two-step method to improve the separation of small micronuclei (MN) from the main nuclei for flow cytometry-based MNT presented by Nüsse and Kramer (17). However, consistently the same problems were encountered: (i) lack of a reliable proliferation assay to show comparable sensitivity to the Cytochalasin B (Cyto B) MNT required for the primary cells and (ii) the inability to differentiate and ultimately exclude DNA originating from apoptotic and necrotic cells that may contaminate the MN area. Since mitosis is a prerequisite for expression of MN, it is compulsory to demonstrate that cell division occurred and if possible to identify the cells that completed mitosis. Since primary HuLy do not respond equally to mitogen stimulation, a reliable proliferation marker is required. Currently, the ‘gold standard’ for the HuLy MNT assay is the use of the Cyto B (2,19). Cyto B is an inhibitor of actin polymerisation that induces cytokinesis block (2,19,20) and, therefore, enables proliferation monitoring. Indeed, cells that have not divided in culture will appear as mononucleated cells, cells that divided once will be binucleated and the cells that divided two or more times will be polynucleated (19). As a consequence, the cytotoxicity assessment and the MN induction can be analysed simultaneously. As a flow cytometry-based proliferation assay, bromodeoxyuridine (BrdU) incorporation has been used for the HuLy MNT (21). BrdU incorporation combined with the Hoechst quenching (22) or the fluorescent anti-BrdU antibody binding (23–25) enables the discrimination between cells that did not divide, divided once, twice or more. However, BrdU is genotoxic (23–26) and, therefore, may interfere with the potential genotoxic effect of the test compound. Another marker used for lymphocyte division and tracking in vivo and up to date not used as a proliferation marker for MNT is carboxyfluorescein succinimidyl ester (CFSE) (27,28). CFSE is a fluorescent product of carboxyfluorescein diacetate succinimidyl ester (CFDA-SE). CFDA-SE is a non-fluorescent compound that diffuses through cell membranes and is cleaved by non-specific cellular esterase forming CFSE that binds to cellular proteins with a special affinity to lysine. CFSE is equally divided between daughter cells upon cell division and enables cell fate tracking up to eight divisions (27,28). The second problem in the context of the flow cytometry technology is the discrimination of DNA particles originating from apoptotic or necrotic cells may interfere with the flow cytometric assessment of MN frequency. In this context, a technique presented by Viaggi et al. (15) showed a significant improvement. Incorporation of antibody conjugated (anti-CD2) magnetic separation of analysed cells prior to staining and lysis procedure significantly reduced the number of debris of apoptotic and necrotic origin that interfered with the MN gating area. Another solution was the staining suggested by Avlasevich et al. (9) and further developed and improved by Bryce et al. (10,11,29). The method successfully reduces the number of apoptotic bodies and necrotic cells from the MN scoring region. The assay is based on the sequential staining of DNA fragments originating from late apoptotic and necrotic cells with ethidium monoazide (EMA) (30). The work presented in this manuscript was initiated to develop a novel flow cytometry-based primary HuLy MNT method. This new assay is based on a three-step staining procedure: CFSE as a proliferation marker, EMA for chromatin of necrotic and late apoptotic cells discrimination (9–11) and 4,6-diaminodino-2-phenylindole (DAPI) (31,32) as a DNA marker. The proof-of-principle of the new flow cytometry-based method was performed by comparing the MNT results obtained from the triple staining flow cytometry analysis and Cytochalasin B/Giemsa microscopy analysis. Methyl methanesulfonate (MMS), mitomycin C (MMC), vinblastine sulphate (VB) and cyclophosphamide (CP) were included in the study as genotoxic compounds; sodium chloride (NaCl) as a non-genotoxin, dexamethasone (DEXA) as an apoptosis inducer. Materials and methods Cells and culture medium Blood was obtained from seven different healthy, non-smoking donors aged between 20 and 47 years. Peripheral blood mononuclear cells (PBMCs) were isolated using Vacutainer CPT tubes (Becton Dickinson, Franklin Lakes, NJ, USA), 8 ml of whole blood per Vacutainer CPT tube. The cultures were prepared using blood from a single donor per experiment. Blood from a different donor was used for the repeat experiment. The isolated PBMCs were washed three times with phosphate-buffered saline (PBS) (Oxoid, Hampshire, England). The cultures for microscopy evaluation (HuLy Cyto B) were prepared in duplicates in 12-well plates. For flow cytometry-based MNT analysis, PBMCs in a final density of 50 × 106 cells/ml were stained prior to cultivation for 10 min at room temperature with 5 μM CFDA-SE purchased from Fluka (Schnelldorf, Germany) and washed three times with PBS (Oxoid) with 5% foetal bovine serum (FBS) (Invitrogen, Merelbeke, Belgium) before cultivation. In order to determine the intensity of CFDA-SE staining, two sets of controls were prepared: (i) cellular autofluorescence and (ii) non-divided cells. For the cellular autofluorescence control, PBMCs were cultivated after isolation and washing step, omitting the CFDA-SE staining. In order to position the peak of non-divided cells, a culture of non-stimulated PBMCs stained with CFDA-SE was prepared (culture medium without phytohemaglutinin) (Figure 1). Fig. 1 View largeDownload slide The CFSE staining control. Auto fluorescence: cells were without the CFDA-SE staining and express the background level of the cells fluorescence. Non-divided: cells were cultured in the absence of phytohemaglutinin in order to position the peak of non-divided cells. Negative control: the peaks presented in the graph negative control represent the sequential divisions appearing in the stimulated cultures with no changes performed after positioning the non-divided peak. Fig. 1 View largeDownload slide The CFSE staining control. Auto fluorescence: cells were without the CFDA-SE staining and express the background level of the cells fluorescence. Non-divided: cells were cultured in the absence of phytohemaglutinin in order to position the peak of non-divided cells. Negative control: the peaks presented in the graph negative control represent the sequential divisions appearing in the stimulated cultures with no changes performed after positioning the non-divided peak. The HuLy cultures at a concentration of 0.7 × 106 cells/ml were incubated in 37°C in humid atmosphere and with 5% CO2. The culture medium consisted of RPMI 1640 + GlutaMAX I + 25 mM HEPES (Invitrogen) supplemented with 15% (v/v) of heat inactivated FBS (Invitrogen) and 1%(v/v) penicillin-streptomycin (Invitrogen) and 3.75% phytohaemagglutinin (Invitrogen). Rat liver S9 microsomal fraction was used for metabolic activation (10% S9 mixture in final culture) for CP. S9 mixture was prepared in distilled water and contained: 10% of S9 microsomal fraction (Trinova Biochem Gmbh, Giessen, Germany), glucose-6-phosphate 1.25 mg/ml (CAS. 3671-99-6; Roche Diagnostics, Mannheim, Germany), NADP, disodium salt 0.53 mg/ml (CAS.24292-60-2; Merck, Darmstadt, Germany), potassium chloride 2.24 mg/ml (CAS. 7447-40-7; Merck), sodium hydrogen carbonate 1.5 μg/ml (CAS. 144-55-8; Merck), 10% Hanks Balanced Salt Solution 10× (Gibco, Invitrogen, Auckland, New Zealand). All cell cultures (for microscopy evaluation and for flow cytometry samples) were cultivated for 68 h. The treatment started 24 h after the culture initiation. The samples for microscopy evaluation (HuLy Cyto B) were treated with 6 μg/ml Cytochalasin B at 44 h after the culture initiation. Compounds and treatment schedules As recommended by the latest ‘OECD Guideline for the Testing of Chemicals—In Vitro Mammalian Cell Micronucleus Test 487’—Annex 3 (2), the selection of compounds was: VB (CAS. 143-67-9; Sigma, Schnelldorf, Germany), MMC (CAS. 50-07-7; Sigma), MMS (CAS. 66-27-3; Fluka), CP (CAS. 50-18-0; Baxter AG, Volketswil, Switzerland), sodium chloride (CAS. 7647-14-5; Merck), DEXA (CAS. 50-02-2; Sigma). Treatment started 24 h after culture initiation and lasted for 44 h with no recovery time. CP treatment was performed 24 h after culture start and lasted for 3 h with S9 mix followed by 41-h recovery time. Cytokinesis-block HuLy MNT (microscopy evaluation) For microscopy analysis, duplicate slides were prepared at harvest for each culture using cytospin technique (Cytospin 3, Shandon, Hampshire, England). Air-dried slides were fixed for 10 min using 100% methanol. Fixed and air-dried slides were placed for 30 min in previously filtered 2.6% solution of Giemsa azur eosin methylene blue solution (Merck) in WEISE Buffer solution (previously prepared using WEISE Buffer tablets pH 7.2; Merck). One thousand T-lymphocytes for cytotoxicity assessment and 2000 binucleated T-lymphocytes for MN frequency per culture were analysed. Duplicate cultures per concentration were prepared. Flow cytometry-based HuLy MNT The flow cytometry analysis was performed with an FACS LSR II (with Diva 6.1.2 software) cytometer equipped with argon (488 nm) and ultraviolet (355 nm) lasers. The cytometer uses the pulse analysis system, capable of providing a height, width and area value for each parameter. The height (H) value reflects the intensity of the analysed particle fluorescence, the width (W) represents the time required for the analysed particle to progress through the laser beam and the area (A) is the parameter that describes the intensity of the analysed particle fluorescence expressed in a timely manner. At harvest, cell cultures were transferred into the 15 ml centrifuge tubes and centrifuged at 600g for 5 min. The supernatant was removed and the cells were resuspended by gentle tube taping. Three hundred microlitres of EMA solution (0.125 mg/ml in PBS with 2% of FBS) was added to the cell suspension. For the photo-activation step, tubes were placed on ice under the visible light source for 25 min. After the photo-activation step [EMA covalently binds the DNA of cells with disrupted membrane integrity—dead/dying cells (9–11,33)], the tubes were protected from light exposure for the remaining steps of the staining procedure. To each tube, 5 ml of cold buffer (PBS with 2% FBS) was added and the cells were centrifuged with 600g for 5 min. The supernatant was discarded and the cells were resuspended by gentle tube taping. The cells suspension was kept in room temperature before cell lysis. The cells were lysed in a two-step procedure [a modified procedure described by Nüsse and Kramer (17) and Avlasevich et al. (9–11)]. The lysis solution 1 (0.5 ml) was added to the tubes and vortexed. Lysis solution 1 consisted of 0.584 mg/ml NaCl (CAS. 7647-14-5; Merck), 1 mg/ml sodium citrate (CAS. 6132-04-3; Sigma), 0.3 μg/ml IGEPAL-630 (CAS. 9036-19-5; Fluka), 250 μg/ml RNase A (Qiagen, Hilden, Germany) 1 μg/ml DAPI (CAS. 28718-90-3; Sigma). The samples were incubated at room temperature for 1.5 h. At the end of the incubation, 0.5 ml of lysis solution 2 was added into the samples and immediately vortexed. Lysis solution 2 consisted of 85.6 mg/ml sucrose (CAS. 57-50-1; Sigma), 15 mg/ml citric acid (CAS. 5949-29-1; Merck) and 1 μg/ml of DAPI (CAS. 28718-90-3; Sigma). Since the CFSE fluorescence proved to be pH dependent (34), the pH of the lysis solution 2 was adjusted to 8–10. The specimens were measured immediately after the staining. At least 30 000 gated, divided nuclei (EMA negative, DAPI positive, CFSE indicating cell division) per sample were analysed (two samples per culture, two cultures per concentration). The template for analysis uses several parameters, including forward scatter, side scatter, DAPI and EMA fluorescence, to exclude potential contamination of MN and nucleus region with false events like late apoptotic/necrotic chromatin or debris. The detailed gating strategy is presented in Figure 2. Fig. 2 View largeDownload slide Gating strategy based on FSC, SSC, DAPI fluorescence, EMA fluorescence and CFSE fluorescence. Plot A (FSC-A versus SSC-A)—discrimination based on size (FSC) and granularity (SSC), the beads gate is used for calibration of the experiment; Plot B (DAPI-W versus DAPI-A)—excludes the events that show an increased value of the DAPI fluorescence; Plot C (histogram on DAPI-H)—excludes the events that show DAPI fluorescence <1/100 of the G1 population; Plot D (DAPI-A versus FSC-A)—the FSC versus DAPI gate discrimination-based size and the fluorescence of 1/100 of the G1 population; Plot E (DAPI-A versus SSC-A)—the SSC versus DAPI gate includes all nuclei and all events that show the granularity and the fluorescence of 1/100 of the G1 population; Plot F (EMA-A versus DAPI -A)—the EMA-positive and EMA-negative gating strategy enables exclusion of the apoptotic/necrotic events; Plot G (DAPI-A versus FSC-A)—events that fulfil all gating conditions described above are classified for plot G, where nuclei and MN are presented. MNs were defined as events between 1/100th and 1/10th of DAPI-associated fluorescence of the main 2n nuclei; Plot H (histogram CFSE-H)—nuclei accepted in nuclei gate were analysed for proliferation status (reduced CFSE fluorescence); Plot H (histogram CFSE-H)—nuclei accepted in nuclei gate were analysed for proliferation status (reduced CFSE fluorescence); Plot I (Histogram DAPI-A)—additionally, nuclei accepted in final classification may be analysed for DNA content using DAPI fluorescence. Fig. 2 View largeDownload slide Gating strategy based on FSC, SSC, DAPI fluorescence, EMA fluorescence and CFSE fluorescence. Plot A (FSC-A versus SSC-A)—discrimination based on size (FSC) and granularity (SSC), the beads gate is used for calibration of the experiment; Plot B (DAPI-W versus DAPI-A)—excludes the events that show an increased value of the DAPI fluorescence; Plot C (histogram on DAPI-H)—excludes the events that show DAPI fluorescence <1/100 of the G1 population; Plot D (DAPI-A versus FSC-A)—the FSC versus DAPI gate discrimination-based size and the fluorescence of 1/100 of the G1 population; Plot E (DAPI-A versus SSC-A)—the SSC versus DAPI gate includes all nuclei and all events that show the granularity and the fluorescence of 1/100 of the G1 population; Plot F (EMA-A versus DAPI -A)—the EMA-positive and EMA-negative gating strategy enables exclusion of the apoptotic/necrotic events; Plot G (DAPI-A versus FSC-A)—events that fulfil all gating conditions described above are classified for plot G, where nuclei and MN are presented. MNs were defined as events between 1/100th and 1/10th of DAPI-associated fluorescence of the main 2n nuclei; Plot H (histogram CFSE-H)—nuclei accepted in nuclei gate were analysed for proliferation status (reduced CFSE fluorescence); Plot H (histogram CFSE-H)—nuclei accepted in nuclei gate were analysed for proliferation status (reduced CFSE fluorescence); Plot I (Histogram DAPI-A)—additionally, nuclei accepted in final classification may be analysed for DNA content using DAPI fluorescence. On the plots (A–I), a series of gating strategies was applied: Plot A (FSC-A versus SSC-A)—light scatter gate includes events that are 1/100 of the nuclei size (FSC) and 1/100 of the nuclei granularity (SSC), the beads gate is used for calibration of the experiment and the main population of nuclei should show the comparable size and granularity to the beads; Plot B (DAPI-W versus DAPI-A)—the purpose of the ‘doublet discrimination’ gate is to exclude the events that show the increased values of the DAPI fluorescence; Plot C (histogram on DAPI-H) excludes the events that show DAPI fluorescence <1/100 of the G1 population. Plot D (DAPI-A versus FSC-A)—the FSC versus DAPI gate includes all nuclei and all events that show the size and the fluorescence of 1/100 of the G1 population; Plot E (DAPI-A versus SSC-A)—the SSC versus DAPI gate includes all nuclei and all events that show the granularity and the fluorescence of 1/100 of the G1 population; Plot F (EMA-A versus DAPI-A)—the EMA positive and EMA negative gating strategy enables exclusion of the apoptotic/necrotic events; Plot G (DAPI-A versus FSC-A)—events that fulfil all gating conditions described above are classified for plot G where nuclei and MN are presented. MNs were defined as events between 1/100th and 1/10th of DAPI associated fluorescence of the main 2n nuclei; Plot H (histogram CFSE-H)—nuclei accepted in ‘nuclei’ gate were analysed for proliferation status (reduced CFSE fluorescence); Plot I (histogram DAPI-A)—additionally, nuclei accepted in final classification may be analysed for DNA content using DAPI fluorescence. Cytotoxicity assessment As recommended by the ‘ICH S2 (R1) guidance on genotoxicity testing and data interpretation for pharmaceuticals intended for human use’ (3), the top concentration should aim 55 ± 5% of cytotoxicity for the in vitro MN assay. If no toxicity is observed, the top concentration recommended is 1 mM or 0.5 mg/ml whichever is lower (3). In the present study, the 60% cytotoxicity cut-off value according to CytoB replicative index (RI) was used. The RI proposed by Kirsch-Volders et al. (35) and recommended by OECD guideline 487 (2,36,37) was used as a cytoxicity parameter and was calculated as follows:  where, T is treated and C is control. In order to mimic the Cyto B calculations, the CFSE-based results have been calculated as follows. The T lymphocytes that did not divide would appear in Cyto B MNT as mononucleated cells and therefore, the number of nuclei classified with CFSE as non-divided was divided by factor 1 (A). The T lymphocytes that divided once in Cyto B MNT would appear as binucleated cells and therefore, the number of nuclei classified with CFSE as 1 division are divided by factor 2 (B). Since in Cyto B MNT, polynucleated cells would contain three or more nuclei the number of nuclei classified with CFSE as 2 divisions was divided by value 4 (C), 3 divisions was divided by factor 8 (D) and 4 divisions are divided by factor 16 (E). The final RI for flow cytometry was calculated as follows:  A = Number of nuclei (0 division)/1. B = Number of nuclei (1 division)/2. C = Number of nuclei (2 divisions)/4. D = Number of nuclei (3 divisions)/8. E = Number of nuclei (4 divisions)/16. EMA-positive events The frequency of EMA-positive events was calculated as the ratio of total EMA-positive, DAPI-positive events to divided nuclei (EMA negative, DAPI positive). The results were expressed as fold increase of negative control. Correlation coefficient The correlation coefficient (R2) between dataset values was calculated using the Microsoft Office Excel 2007 software. MN induction and RI values obtained with microscopy and flow cytometry were compared. Caspase 3/7 detection Vybrant FAM CASPASE 3/7 assay kit has been purchased from Molecular Probes (Invitrogen). The providers’ protocols have been followed. Briefly, the assay is based on fluorescent inhibitor (FLICA) of caspase 3 and 7. The reagent associates a fluoromethyl ketone (FMK) moiety, which can react covalently with a cysteine to form aspartic acid–glutamic acidvaline–aspartic acid (DEVD). The FLICA reagent is thought to interact with the enzymatic reactive centre of an activated caspase via the recognition sequence and then to attach covalently through the FMK moiety. Terminal deoxynucleotide transferase deoxyuridine triphosphate nick end labelling FragEL DNA Fragmentation Detection Kit (Fluorescent-TdT Enzyme) was purchased from Calbiochem (Darmstadt, Germany). The providers’ protocol has been followed. Briefly, FragEL DNA Fragmentation Detection Kit labels hydroxyl groups generated during the apoptotic endonucleases DNA degradation with fluorescein-conjugated deoxynucleotides. Experiment validity criteria For both methods (microscopy and flow cytometry), experiments were considered valid if: the MN frequency of the solvent control was within the range of the solvent control historical values (microscopy: MN% = 0.35 ± 0.1, n = 14 cultures; flow cytometry MN% = 0.42 ± 0.16, n = 26 cultures) the MN frequency of the positive control (MMC 0.03 μg/ml) was within the range of positive control historical values (microscopy: MN% = 2.23 ± 0.39, n = 10 cultures; flow cytometry MN% = 2.91 ± 0.79, n = 12 cultures). Positivity criteria for MN test For both methods (microscopy and flow cytometry), a compound was considered positive for MN induction, if it induced a reproducible increase in the MN frequency according to the following criteria: the frequency of MN showed at least a double increase over the concurrent solvent control the MN frequency and was above the maximum value of the historical negative control range. Results The ‘proof-of principle’ of the flow cytometry-based primary HuLy MNT was done by comparing the flow cytometry results with the traditional Cyto B MNT using the following genotoxic compounds: MMC, VIN, MMS, CP(+S9); NaCl a non-genotoxic compound and the apoptosis inducer DEXA. The flow cytometry CFSE-based RI assessment showed a very good positive correlation with Cyto B-based RI microscopy results for all tested compounds (Table I). The calculated values of the correlation coefficient between the datasets (R2) are presented in the Table I. For MMC, MMS, CP, NaCl and DEXA, R2 values were close to 0.9. A lower correlation coefficient value (R2 = 0.61) was observed for VB, however, the compound showed no cytotoxicity up to the highest tested concentration. Therefore, even small variations may result in a decrease of the R2 values. Table I Correlation coefficient (R2) of the RI and MN frequency obtained by flow cytometry and CytoB microscopy scoring Compound  MNT  RI  R2  R2  MMS  0.744  0.97  MMC  0.97  0.99  VB  0.99  0.62  CP  0.65  0.92  NaCl  0.98  0.86  DEXA  0.98  0.89  Compound  MNT  RI  R2  R2  MMS  0.744  0.97  MMC  0.97  0.99  VB  0.99  0.62  CP  0.65  0.92  NaCl  0.98  0.86  DEXA  0.98  0.89  View Large A concentration-dependent increase in MN frequency was observed with all tested genotoxic compounds using both flow cytometry and microscopy analysis (Table I, Figure 3). Fig. 3 View largeDownload slide Cytotoxicity and MN induction after treatment with: MMC, VB, MMS, NaCl and DEXA. All results for RI and MN are mean values of replicate cultures ± standard deviation (error bars). *positive result in MN frequency (according to fold increase positivity criteria); micr, microscopy. Fig. 3 View largeDownload slide Cytotoxicity and MN induction after treatment with: MMC, VB, MMS, NaCl and DEXA. All results for RI and MN are mean values of replicate cultures ± standard deviation (error bars). *positive result in MN frequency (according to fold increase positivity criteria); micr, microscopy. A concentration-dependent increase was observed for MMC and VB with both systems (Figure 3). Flow cytometry results correlated positively with the Cyto B MNT (MMC R2 = 0.97, VB R2 = 0.99). A lower R2 value (R2 = 0.74) was observed for MMS, where in the highest analysed concentrations (25 μg/ml), the MN frequency was higher in the flow cytometry assay in comparison to microscopy (Figure 3). The larger difference in MN frequency between microscopy and flow cytometry appears, though only at borderline cytotoxicity (microscopy RI = 40.9%; flow cytometry RI = 38.3%). A small discrepancy in flow cytometry and microscopy MN frequency results were noted after CP treatment (R2 = 0.65). The MN frequency obtained by flow cytometry was lower than the MN frequency scored with Cyto B MNT. Despite this discrepancy, the positive effects were reached at the same concentration (Figure 3). As expected, no significant MN frequency increase was observed in NaCl-treated cultures (Figure 3) using either flow cytometry or microscopy analysis. No MN induction was recorded after treatment with DEXA using both analysis systems (Figure 3). This result shows that only an insignificant number of apoptotic bodies interfered with the MN scoring, after staining with EMA even when the toxicity was exceeding 40% of RI. In order to confirm the induction of apoptosis by DEXA and confirm the role of EMA in the apoptotic bodies exclusion, follow-up tests were performed using: Caspase3/7, TUNEL and EMA-positive events analysis. A clear induction of apoptosis was observed with all three test systems. Additionally, a comparison of the flow cytometry-based HuLy MNT in the presence of (+EMA) and in the absence of EMA (−EMA) was performed. Inclusion of EMA in the staining procedure resulted in a successful exclusion of the apoptotic bodies from MN gating region (+EMA), whereas in the case of −EMA, a clear dose-dependent MN induction effect was observed after DEXA treatment (Figure 4). Fig. 4 View largeDownload slide Apoptosis and MN induction after treatment with DEXA. Analysis performed including EMA (+EMA) staining and alternate staining procedure with no EMA incorporation (−EMA) together with apoptosis induction, analysed with Caspase 3 and 7 level (Caspase 3/7), TUNEL and EMA events increase (EMA) as an increase over current solvent control. Fig. 4 View largeDownload slide Apoptosis and MN induction after treatment with DEXA. Analysis performed including EMA (+EMA) staining and alternate staining procedure with no EMA incorporation (−EMA) together with apoptosis induction, analysed with Caspase 3 and 7 level (Caspase 3/7), TUNEL and EMA events increase (EMA) as an increase over current solvent control. To investigate the reproducibility of the assay, 15 independent experiments with negative control (vehicle) and positive control (MMC 0.03 μg/ml) treated cultures were performed using blood from seven different donors (Figure 5). The data show a high level of inter-experimental as well as inter-donor reproducibility. Fig. 5 View largeDownload slide Inter-experimental and inter-individual variability of MN frequencies in vehicle-treated cultures (negative controls: Plot A) and MMC (0.03 μg/ml)-treated cultures (positive controls: Plot B) reproducibility data. Fifteen independent experiments were performed using blood from seven different donors. The error bars—standard deviation for replicate cultures for one donor; the solid line—the mean value for MN frequency for all donors and the dash line—the mean value for all donors ± standard deviation. Fig. 5 View largeDownload slide Inter-experimental and inter-individual variability of MN frequencies in vehicle-treated cultures (negative controls: Plot A) and MMC (0.03 μg/ml)-treated cultures (positive controls: Plot B) reproducibility data. Fifteen independent experiments were performed using blood from seven different donors. The error bars—standard deviation for replicate cultures for one donor; the solid line—the mean value for MN frequency for all donors and the dash line—the mean value for all donors ± standard deviation. Discussion The purpose of the study presented in this manuscript was to establish a new flow cytometry-based primary HuLy MNT assay where conveniently all parameters (RI, MN frequency taken into account apoptosis) can be measured in one flow cytometric assessment. The main challenges for the method were to develop a reliable and a sensitive cell proliferation marker and the reduction of artificially positive results caused by interference of DNA originating from dead and dying cells with the MN region. Proliferation marker Since mitosis is a prerequisite for expression of MN, it is compulsory to demonstrate that cell division has occurred. The primary HuLy response to the mitogen stimulation varies between culture conditions and donors. The use of Cyto B or another proliferation approach is therefore mandatory for division monitoring for primary HuLy MNT (2,38). The effect of a test substance on cell proliferation may be monitored in parallel cultures, however, more conveniently should be scored within the same measurement as the MN frequency (2). In the present work, we introduced CFSE as a new dye for cell proliferation monitoring. CFDA-SE is a non-fluorescent compound that diffuses through cell membranes and is cleaved by non-specific cellular esterase to CFSE, a fluorescent product that binds to cellular proteins with a special affinity to lysine (27,28). CFSE is equally divided between daughter cells upon cell division and enables cell fate tracking up to 8 divisions. The assay is successful in cell division determination in in vitro and in vivo studies (28). Additionally, the halving effect of the CFSE fluorescence is retained in the nuclei (27,39). CFSE shows pH-dependent fluorescence emission upon 488-nm laser exposure, its fluorescence increases with the increase in pH value. This fact is correlated with the formation of the fluorescent mono- and di-anion form of CFSE (34). During the assay development, three main questions had to be addressed: (i) Halving of the nuclear CFSE fluorescence of primary human lymphocyte T after each cell division, (ii) absence of CFSE fluorescence interference after treatment with various test compounds and (iii) no interference in MN separation is observed after the lysis solution pH adjustments. Our data show that the fluorescence is decreased by a factor of 2 after each cell division and the results of CFSE-based RI are in a very good positive correlation with Cyto B method used as a reference. No interference of the tested compounds with the CFSE fluorescence was observed. In our study, the two-step lysis procedure was applied (9–11,17,29,40) after modification. A low pH value of the solutions blocked the fluorescence of the CFSE, therefore, the pH of the solution had to be adjusted to 8–10. The pH adjustments did not decrease the separation of MN from nuclei and a positive correlation has been observed for MN frequencies scored by flow cytometry and microscopy. With CFSE-based cytotoxicity assessment, we were able to track the exact number of divisions for each nucleus. The BrdU methodology on the contrary enables only differentiation of non-proliferating cells from cells up to two subsequent cell cycles (41). In addition, CFSE proved to be non-genotoxic (data not shown) and therefore, no interaction with the effects of the test compound is expected. On the contrary, BrdU has the potential to induce numerical or structural chromosome alterations and, therefore, may influence the test outcome (25,26). Apoptosis/necrosis aspect Another known confounding factor for the flow cytometry-based MNT is the potential interference of necrotic and apoptotic events with the determination of MN frequency, which may have an influence on the genotoxicity assessment of a tested compound and could lead to false-positive results (16–18,40). A significant improvement in the separation of apoptotic and necrotic human T lymphocytes was achieved by the incorporation of magnetic beads separation (15). We used EMA, a DNA dye, specific for late apoptotic and necrotic cells (9–11,29), which proved to be successful and showed a very good positive correlation with traditional microscopy results. Avlasevich et al. (9) and Bryce et al. (11) showed no MN induction after DEXA or staurosporin treatments of L5178Y cells scored by flow cytometry providing EMA staining was applied. In our study, the use of EMA staining to exclude late apoptotic/necrotic events has been successful in the discrimination of dead and dying cells’ DNA. A well-known apoptogenic compound, DEXA, showed no MN induction potential up to 33% RI providing EMA staining was included. However, in the absence of EMA staining, DEXA treatment induced a concentration-dependent increase in MN frequency. A significant difference in MN frequency has been recorded between the flow cytometric and the microscopic MN assessment for the highest concentration of MMS (25 μg/ml). The discrepancy has already been reported in our previous study (42) for TK6 cell line. We speculated that, since EMA is a membrane impermeable fluorescent dye, cells in mid and early apoptosis stages may not be detected with this staining and therefore, DNA of this origin may interfere with the MN region. This explanation is, however, contradictory to our data and the data obtained by Bryce et al. (11), which proved no MN increase after Staurosporin or DEXA treatment. Hence, it may be speculated that high rate of MN events scored in flow cytometry at highest tested MMS concentration may in fact be a compound-specific response and is probably correlated with extremely high cytotoxicity. Moreover, our data prove that the negative control-treated (vehicle) and positive control (MMC 0.03 μg/ml)-treated cultures show reproducible MN frequency among different donors and different experiments. In conclusion, we have shown that the new flow cytometry-based primary human T-lymphocyte MNT method is at least equally reliable as the standard Cytochalasin B MNT and has the potential for further culture miniaturisation and full automation. Being faster and less tedious than the microscopic analysis, the flow cytometry-based methodology showed very promising results. Conveniently, the division tracking is scored within the same sample as the MN frequency. 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TI - In vitro primary human lymphocyte flow cytometry based micronucleus assay: simultaneous assessment of cell proliferation, apoptosis and MN frequency
JF - Mutagenesis
DO - 10.1093/mutage/ger044
DA - 2011-07-26
UR - https://www.deepdyve.com/lp/oxford-university-press/in-vitro-primary-human-lymphocyte-flow-cytometry-based-micronucleus-ncH0Z4BrFW
SP - 763
EP - 770
VL - 26
IS - 6
DP - DeepDyve
ER -