High-throughput and high content micronucleus assay in CHO-K1 cells

Abstract

Visualization of micronuclei induction by chemicals and drugs enables measurement of possible compound genotoxicity. A loss of entire chromosome or a fragment of chromosome can lead to formation of micronuclei (MNi). The in vitro micronucleus assay can be conducted using nuclear dyes with high content imaging platforms. This chapter describes the cytochalasin block method of measuring micronuclei in CHO-K1 cell lines.

1. Introduction:

The study of DNA damage at chromosomal level is a vital part of genetic toxicology. DNA damage may result from chromosome loss or from mal-segregation of a chromosome, leading to an important event in aging and carcinogenesis [1]. Chromosome loss and mal-segregation are probably caused by defects in a spindle or a centromere, but they may be a consequence of under-condensation of a chromosome structure before metaphase [2–4]. Evaluation of chromosome aberrations in metaphases is often used in the cytogenetic research [5]. Although this approach provides the most detailed analysis, the complexity and laboriousness of this method, in addition to the artefactual loss of chromosomes from metaphase preparations, call for the development of simpler systems to measure chromosomal damage [1].

Heddle in 1973 [6] and Schmid in 1975 [7] independently proposed measurement of micronuclei (MNi) in vivo in dividing cell populations, such as the bone marrow and peripheral blood erythrocytes, as an alternative and simpler method of assessing genotoxicity that was also more robust [6–8]. Although these assays are now one of the best established in vivo cytogenetic assays in the field of genotoxicity, it has limited applicability to other cell types. To overcome this limitation, Fenech et al. developed a method that can measure MNi in a variety of nucleated cells [1]. MNi are ideally scored in the binucleated stage of cells, as MNi are expressed in cells that are completing nuclear division [9,10]. In addition, several methods based on stathmokinesis, flow cytometry, and DNA labeling approaches have been developed. Among these methods, cytokinesis-block micronucleus (CBMN) method is found to be most favored in vitro micronucleus assay (IVMN) because of its ease, speed, reproducibility, and ability to concurrently estimate mitotic delay and lack of uncertainty regarding its effect on baseline genetic damage [9–12]. So far, CBMN has been proven to be an effective tool for the study of cellular and nuclear dysfunction caused by aging, deficiency or excess of micronutrient, genotoxin exposure and genome maintenance [13]. This method is also helpful in the emerging fields of nutrigenomics and toxicogenomics because nutrient status may affect the sensitivity of the cells to exogenous genotoxins [14].

For many years being a part of several recommended regulatory battery tests for testing genotoxicity, in vitro micronucleus assay is routinely used as a rapid screening test [15]. For example, OECD guidelines require that the micronuclei frequency be measured in binucleated cells [16]. CBMN is a very useful approach of detecting micronuclei in binucleated cells, making this method an excellent candidate for automated image-analysis [12]. Various high-content systems have been developed using several automated scoring methodologies owing to faster delivery of results and reduced variability among scorers [17]. The experiment procedure is the same for both manual and automated scoring. In manual scoring, a trained operator reads the slides under a microscope. Automated scoring, on the other hand uses proprietary image analysis software specially designed for the particular imaging system [18].

Recent publications indicate that the automated IVMN assay on cultured cells is a powerful genotoxicity assay with cellular imaging [18,19]. In 2013, Tilmant et al., using CHO-k1 cells in automated micronucleus assay, found 91% predictivity with a sensitivity of 94% and a specificity of 85% [20]. Here, we describe the CBMN- based method of micronuclei detection using automated high- content imaging analysis coupled with fluorescent microscopy in 384-well plate format in CHO-K1 cell line [21,22].

2. Materials

Prepare all solutions using ultrapure water (prepared by purifying deionized water to attain a sensitivity of 18 MΩ cm at 25 °C) and analytical grade reagents. Prepare all solutions at room temperature unless indicated otherwise.

2.1. Sample preparation

1. Chinese hamster ovary (CHO-K1) cells.

2. Culture medium (CM): F-12K Nutrient Mixture supplemented with 10% FBS and 100 U/mL penicillin and 100 μg/mL streptomycin. Store at 4 °C.

3. Collagen I coated 384-well black wall/clear bottom plate.

4. Positive control compounds: cyclophosphamide (CP) (CASRN (Chemical Abstract Services Registry Number = 6055–19-2), mitomycin C (MMC) (CASRN = 50–07-7). Staurosporine (CASRN =62996–74-1)

5. 1 mg/ml Cytochalasin B

6. Ultrapure distilled water

7. 20 % Aroclor 1254-induced Sprague Dawley male rat liver S9 mix (MUTAMYME ^™ S9 Mix, Moltox)

2.2. Fluorescent staining and imaging

• 1.Hank’s balanced salt solution (HBSS).
• 2.Fixing solution: 8% Paraformaldehyde (from 32 % stock) and 0.2% Hoechst 33342 (from 10 mg/ml stock) 0.04% Red Cell Mask (from 10 mg/ml stock) and 0.4% Cell Event^™ Caspase-3/7 Green Detection Reagent (2 mM stock) in HBSS.
• 3.384-well plate sealing film.
• 4.Pipettes
• 6.8-channel aspirator
• 7.ImageXpress Micro Widefield High- Content Screening System (Molecular Devices)

3. Methods

Carry out all procedures at room temperature (RT) unless otherwise specified.

3.1. Compound treatment

1. Cell dispensing: Calculate the cells preferably in single cell suspension by using the cell strainer and adjust the cell density for +S9 and −S9 conditions. Plate 4500 cells/well/25μl for +S9 condition or 750 cells/well/25μl for -S9 condition into collagen I coated 384-well black wall/clear bottom plate and incubate the assay plates for 4 hours to allow proper attachement of cells to the well at 37 °C under a humidified atmosphere and 5% CO_2..

   Getting a single cell suspension is critical for imaging assays as it facilitates identification of individual cell and then normalizing data accordingly.

   In +S9 condition the cell density is higher than in −S9 condition as per OECD guidelines to combat the probable S9 related toxicity.

2. After 4 hours incubation, add 25 μL/well of the compound (e.g. MMC and CP) with or without 4% S9 on top at desired concentrations into the wells and incubate at 37 °C for 24 h or time optimized for cell line of interest.

   Prepare the stock solutions of 1.2 mM mitomycin C (MMC) and 30 mM cyclophosphamide (CP) in ultrapure distilled water, and 40 mM staurosporin in dimethyl sulfoxide (DMSO). Stock solutions of MMC and CP are stored at −80°C and staurosporin is stored at −20°C. MMC is used as positive control for −S9 condition, CP is used as positive control for +S9 condition and staurosporin is used as positive control for apoptosis. Using apoptotic marker aids in identification of healthy binucleated cells.

   Prepare 20 % S9 by adding 10 ml of ice-cold, sterile, purified water to MUTAZYME ^™ Aroclor 1254-induced Sprague Dawley male rat liver S9 lyophilized with NADPH-regenerating system cofactors and phosphate buffer.

   S9 is an exogenous metabolic activation system needed by some test compounds to exert their genotoxic effect using in vitro test systems. S9 by itself is toxic to the cells and the amount of S9 used in an assay needs to be optimized. Also the above S9 is lyophilized with NADPH-regenerating system cofactors and phosphate buffer at the optimal concentrtaions. Two percent of S9 in the final assay volume is optimal with less cytotoxic effect to the cells.

   Prepare 2× final concentrations of compounds and add into assay plate to get 1× final concentration in assay plate

   S9 mix: Prepare 4 % S9 by adding 2 ml stock of 20% S9 in 8 ml CM.

   MMC: Prepare 2.4 μM (800 ng/ml) (final concentration, 400 ng/ml) MMC by diluting 1.2 mM (400 μg/ml) stock of MMC to intermittent 60 μM MMC by adding 10 μl of stock MMC to 190 μl of 4% S9 mix (for +S9) or CM (for −S9). Then, add 10 μl of 60 μM MMC to 240 μl of 4% S9 mix (for +S9) or CM (for −S9)

   CP: Prepare 71.6 μM (final concentration, 35.8 μM) CP by diluting 30 mM stock of CP to intermittent 1.5 mM CP by adding 10 μl of stock CP to 190 μl of 4% S9 mix (for +S9) or CM (for −S9). Then add 10 μl of 1.5 mM CP to 200 μl of 4% S9 mix (for +S9) or CM (for −S9)

   Staurosporine: Prepare 184 μM (final concentration, 91 μM) staurosporine by diluting 40 mM stock of staurosporine to intermittent 4 μM staurosporine by adding 5 μl of stock to 45 μl of 4% S9 mix (For +S9) or CM (for −S9). Then add 13.5 μl of 4 μM staurosporine to 280 μl of 4% S9 mix (for +S9) or CM (for −S9)

3. After 4 hours incubation, remove compounds with S9, followd by washing 3 times with CM very gently, and then adding 25 μl CM/well. The assay plate is incubated overnight.

   For -S9 condition, the assay plate stays in the incubator for compound treatment overnight.

3.2. Cytochalasin B tretament

1. Remove medium and add Cytochalasin B (final concentration, 3 μg/ml).

   Prepare 3 μg/ml Cytochalasin B by diluting 1 mg/ml stock of Cytochalasin B to intermittent 100 μg/ml Cytochalasin B by adding 100 μl of stock to 900 μl of CM. Then add 600 μl of 100μg/ml Cytochalasin B to 19.4 ml CM.

2. Incubate for 24 hours at 37 °C under a humidified atmosphere and 5% CO₂. (See Note 1)

3.3. Fixation and staining

1. Add 25 μl/well fixing solution on top after 24 h. The various components of fixing solution and the recipe are as follows:

   HCS CellMask: Stock 10 mg/mL HCS CellMask (HCS CellMask^™ Red stain, Life Technologies, Catalog number H32712) - Dissolve the entire content of dye vial in 25 μl DMSO. Store at −20°C.

   HCS CellMask^™ Stains are available in either Blue, Green, Orange, Red, or Deep Red detection spectra. We used HCS CellMask^™ Red stain to label the whole cell to accommodate multiwavelength assay since Hoechst used in the assay is blue and CellEvent Caspase-3/7 is green.

   CellEvent Caspase-3/7: 2 mM stock solution in DMSO (Cell Event^™ Caspase-3/7 Green Detection Reagent, Catalog number C10423, Life Technologies) is used to measure activated caspase 3/7. Although the mechanism by which apoptosis is initiated varies depending on cell type and initiating event, activation of caspases is one of the markers of programmed cell death. The reagent consisting of the DEVD peptide sequence conjugated to a nucleic acid–binding dye acts in a way that the substrate is intrinsically nonfluorescent and only in the presence of activated caspase 3/7, produces a fluorogenic response indicative of apoptosis. The fluorescence emission maximum of the dye is approximately 520 nm. Additionally this no wash protocol preserves fragile apoptotic cells typically lost during wash steps. Lastly, the fluorescent signal from the CellEvent^™ Caspase-3/7 reagent survives fixation and permeabilization, providing the flexibility to perform end-point assays and probe for other proteins of interest using immunocytochemistry. Cell EventTM Caspase-3/7 Green Detection Reagent is used to differentiate the healthy cells from unhealthy cells as required by OECD guidelines for our automated micronucleus assay.

   Add 5.5 mL of 32% paraformaldehyde solution, 4.4 μl of HCS CellMask Red, 44 μl Cell Event Caspase-3/7 green dye and 22 μL of 10 mg/ml Hoechst solution to 16.5 mL of HBSS to make total 22 ml fixing solution.

   Final concentration of paraformaldehyde is 4%, HCS CellMask^™ Red stain is 0.02%, Cell Event^™ Caspase-3/7 Green Detection Reagent is 0.2% and Hoechst is 0.1%.

2. Incubate at RT for 30 min

3. Wash with HBSS two times. During the wash step, be very gentle and careful.

4. Add 50 μl HBSS/well, seal and image or store at 4°C.

3.4. Imaging readout

Acquire images using a 20× objective in ImageXpress Micro Widefield High- Content Screening System using DAPI (acquire nuclear images), Texas Red (acquire whole cell) and FITC (acquire apoptotic cells) filter sets. Acquire at least 1050 binucleated cells per compound treatment using the Micronucleus module, a proprietary analysis protocol with MetaXpress softwere.

3.5. Image Analysis

As described previously [21] the Molecular Devices Systems proprietary MetaExpress Micronucleus analysis module identified stained nuclei using Hoechst based on the size, intensity and distance from adjacent cell. The total number of cells in a well accounted for all the cells in the well. Main nuclei in a well were classified as mononucleated, binucleated, and multinucleated. Micronuclei also stained using Hoechst were identified based on size, intensity and distance from main nuclei. A small nuclear mass attached to main nucleus, was not depicted as micronucleus. The image analysis software provided information on the number of micronuclei in mononucleated, binucleated and multinucleated cells respectively. The percentage of micronuclei in present study represented healthy binucleated cells containing micronuclei. (Figs. 1 and 2)

Fig. 1.

Segmentation in micronucleus analysis module correctly identifies the whole cell, main nucleus and micronucleus by CellMask Red and Hoechst respectively.

Fig. 2.

Identification of micronuclei according to OECD guidelines. Note the red micronuclei identified by a yellow line. Nuclear masses attached to main nuclei; identified by green lines not identified as micronuclei.

3.6. Data Analysis

As described previously [21] the number of micronucleated binucleated cells/1050 binucleated cells in treated cultures was compared with the number of micronucleated binucleated cells/1050 binucleated cells in the corresponding vehicle control culture. Data was expressed as the mean percentage of micronucleated binucleated cells from three replicate cultures ± standard deviation. Statistical significance of the frequency of micronucleated cells in the treated cultures at each dose level compared with the control value was determined using a one-tailed t-test. For cytotoxicity assessment, the nuclear division index (NDI) from MetaXpress software was used. NDI was defined as: (M₁ + 2M₂ + 3M₃ + 4M₄)/N, in which M₁-M₄ represented the number of cells with 1–4 nuclei and N was the total number of viable cells (excluding apoptotic cells). The percentage of cytotoxicity (% Cytotoxicity) was defined as: 100–100{NDI_T −1) / (NDI_D −1)} where, NDI_T = NDI of treated cells; NDI_D = NDI of DMSO control. (Figs. 3 and 4)

Fig. 3.

Induction of micronuclei in CHO-K1 cells. Representative images of nuclei (blue) with micronuclei. Note that without S9 treatment the micronuclei induction increases with higher MMC concentration.

Fig. 4.

Induction of Micronuclei by MMC and CP in CHO-K1 cells. (a) MMC concentration response curves with and without S9 mix. (b) CP concentration response curves with and without S9 mix

4. Notes

Optimization of Cytochalasin-B treatment is very crucial in getting healthy binucleated cells. Also while using vaccum to wash in sample preparation steps, make sure to use slowest possible force to avoid mechanical damange of the sample.