Micronuclei and Comet Assay
To evaluate genotoxic effects of chemical substances in vitro, various methods are available differing in their sensitivity, their practicability, and, finally, the genetic end points considered. The micronucleus assay is one of the methods used to detect chromosomal aberrations in proliferating cell culture systems (Fenech and Morley, 1985). Its advantages are sensitivity as well as uncomplicated realisation and evaluation of results. Because of its relevance and applicability for human cell systems, the cytokinese-block Micronucleus assay with human lymphocytes is described in detail (Fenech, 2000). Micronuclei represent chromosome fragments or whole chromosomes which are not incorporated into the main nuclei at mitosis and they consequently appear only in dividing eukaryotic cells. In order to score micronuclei exclusively in cells that have completed one nuclear division only, the cytokineseblock method is applied in this test version, which prevents the cytoplasmic division after nuclear division by use of cytochalasin B.
The comet assay, or single cell gel electrophoresis, is another widely used assay for identifying genetic damage such as DNA strand breaks. The alkaline version of the assay introduced by Singh et al. (1988) allows the identification of DNA single strand breaks in a very sensitive manner. In addition, there are further advantages, such as the relatively low cell number (<10,000 cells per slide), its ease of application, the applicability of nearly all human and other eukaryotic cells proliferating in vitro, and its time- and costsaving performance, which argue for the use of this assay. To summarize main test principles: agaroseembedded cells are lysed by the help of high salts and detergents; DNA unwinding is promoted by incubating in alkaline electrophoresis buffer (pH > 13); and single-stranded DNA is electrophoresed under alkaline conditions, allowing the DNA fragments to migrate to the anode. After neutralization of slides, the amount of DNA damage may be detected by use of a fluorescent dye such as ethidium bromide visualizing the migrated DNA as the so-called comet tail. The migration length (i.e., the tail length) depending on the DNA fragment size is the most commonly used parameter for quantifying DNA damage. Other metrics considered only by use of image analysis systems are the percentage of migrated DNA and the tail moment (Olive et al., 1990).
Exemplarily, three cell types are presented considering different species (human and rat cells), different culture types (suspension or monolayer culture), and diversity in metabolic activation capacity (normal human bronchial epithelial cells and human lymphocytes versus rat hepatocytes). Selected cell types are capable for genotoxicity testing of single substances and complex mixtures (Mfiller et al., 2002), and the presented test protocols may be transferred to any other eukaryotic cell type proliferating in vitro.
II. MATERIALS AND INSTRUMENTATION
RPMI 1640 instamed (T 121-01) is from Biochrom AG Seromed, as well as the trypan blue solution (L- 6323), phosphate-buffered saline (PBS, L-182-01), collagenase CLS II (C 2-22), fibronectin from human plasma (L 7117), and gentamycine (A-271-23).
Epith-o-ser (FM-56-L) and Leibovitz's L-15 (PM-23-S) are from cc-pro, as well as fetal calf serum (FCS, S-10-L), trypsin-EDTA (Z-26-M), and penicilline-streptomycine solution (Z-13-M). Minimum essential medium Eagle (MEM, M-1018) is from Sigma-Aldrich Chemie, as well as phytohaemagglutinine (PHA, L-9132), cytochalasine B (C-6762), CaCl2 anhydride (C-1016), MgSO4 × 7H2O (M-1880), acridine orange (A-6014), phenol red (P 3532), insulin from bovine pancreas (I-6634), N-[tris(hydroxymethyl) methyl]-2-aminoetharesuforic acid (TES, T 5691), HEPES (H 4034), tricine (T 5816), MgCl2 × 6H2O (24,696-4), Na2HPO4 × 2H2O (21,988-6), and Na2HPO4 × 7H2O (22,199-6) and Na2HPO4 anhydride (S-9763). Normal melting temperative agarose (NMA, 11400) and collagen R (47254.02) are from Serva, as well as ethidium bromide (EtBr, 21251), glucose-6-phospate (22775.01), and Na-pyruvic acid (15220). Low melting temperature agarose (LMA, 35-2010) is from peqLab. Triton X-100 (6683.1), NaCl (3957.2), Na-EDTA (8043.1), Tris (4855.2), dimethyl sulfoxide (DMSO, 7029.1), ethanol (P.076.1), and isopropanole (6752.1) are from Roth. Lymphocyte separation medium (Ficoll-Paque, 17 1440-03) is from Amersham Biosciences. NADP (93208) and formic acid (33015) are from Fluka. Aroclor 1254 (RPC-1254) is provided by Ultra Scientific. All other chemicals are from VWR International: glucose anhydride (1.08337.1000), Na2HCO3 (1.063290.500), methanol (1.06009.1000), glacial acetic acid (1.00056.1000), NaOH (1.06462.1000), KH2PO4 (1.04873.0250), methoxymagresium-methylcarborate (MMC, 8.18156.0100), KCl (1.04933.0500), Na2SO4 (106647), H2O2 (1.08597.1000), 2-mercaptoethanol (1.15433.0050), and L-glutamine (1.00289.0100). Heparin sodium salt (101931) is from ICN Biomedicals Inc. Bovine serum albumin fraction V (11018-025) is from Invitrogen. Peanut oil is from the pharmacy of the clinic of Martin Luther University Halle-Wittenberg.
For preparation of lymphocytes, Leucosep vials (227.290) are used, which are from Greiner. For Aroclor treatment of rats, a gauge needle (Sterican, 4667123) from B. Braun Melsungen AG is used. Surgery instruments are from Sigma Chemie: curved microdissecting forceps (F 4142), dressing tissue forceps (F 4267), straight microdissecting forceps (F 4017), curved microdissecting scissors with a sharp point (S 3271), straight microdissecting scissors with a sharp point (S 3146), scalpel blade (S 2771), and handle (S 2896). Cell culture supplies are from TPP: culture dishes (60cm2, 93100), cell culture vials (20cm2, 91106), cryo vials (2.0ml, 89020), 15-ml centrifuge vials (91015), 50-ml centrifuge vials (91050), serological pipettes (10ml, 94010), and culture flasks (25cm2, 90026). Automatic pipettes (Eppendorf research variable: 0.5-10, 10-100, 20-200, 100-1000, and 500-5000 µl) are from Eppendorf. The pipette aid (Drummond pipette aid, 28081410) is from Heinemann Labortechnik.
Heating plate (453Nl120), water bath (Memmert WB 7, 4623520), swap thermostat (MP 5, 4613411), membrane pump for liquids (ND 100 KT.18, 2245110), electrophoresis chamber (Phero-Sub 2, 344797), power supply (Consors E835, 5822150), and the gel carrier (5817129) are from VWR International. The cell counting chamber (Fuchs-Rosenthal, T731.1), minicentrifuge (X409.1), staining tanks (Hellendahl, H549.1), slides (76 × 26 × 1 mm, 0656.1), and coverslips (24 × 40mm, 1870) are from Roth. For the comet assay, fully frosted slides (61224) from Menzel are used. The CO2 incubation chamber CB 150 (9040-0001) is from wtb-Binder. Clean bench Uniflow (KR-125-GS) is from UniEquip. The table centrifuge (Megafuge, 75003060) is from Kendro. For microscopic analysis, a Nikon fluorescence microscope Eclipse E 600 (MBA 70400) is used. Data processing is performed with the Komet analysis system, including software Komet 4.0 from Kinetik Imaging Ltd., purchased by BFI Optials.
A. Preparation and Cultivation of Cells
All cell types are cultivated in a CO2-incubation chamber under the same conditions: 37°C 5% CO2, and 95% relative humidity.
1. Human Lymphocytes
Heat-inactivated FCS: Fill the necessary volume of FCS in a centrifuge vial, deposit the vial in a water bath, and heat the water continuously up to a temperature of 56°C. Hold this temperature for 30min.
RPMI 1640 cell culture medium: To prepare 1 litre of cell culture medium, add the specified volume of powdered RPMI 1640, 2g NaHCO3, and 750ml dH2O while stirring. Complete to 890ml with dH2O, adjust pH to 7.4, and store at 4°C. Before use, add 100ml of heat-inactivated FCS and 10ml of penicilline-streptomycine and proof the pH to be 7.4.
Phosphate-buffered saline (PBS): To make 1 litre of PBS, add the specified volume of powdered PBS (Ca2+/Mg2+ free) and 750ml dH2O while stirring. Complete to 1 litre with dH2O and store at 4°C
Before starting cell preparation, warm up all media, buffer, and supplements in a water bath (37°C). Use certified buffy coat or other human blood preparation.
2. Hepatocytes of Rat
Presuspension buffer: To make 2 litres, add 1500ml dH2O, 16.6 g NaCl, 1 g KCl, and 4.8 g HEPES while stirring. After dissolving, complete to 2 litres with dH2O and adjust pH to 7.4. Keep at 4°C.
Collagenase buffer: To make 1 litre of stock solution, add 750ml dH2O, 3.9 g NaCl, 0.5 g KCl, 0.7 g CaCl2 × 2 H2O, and 2.4 g HEPES while stirring. After dissolving, complete to 1 litre with dH2O and adjust pH to 7.6. Keep at 4°C. To make 200ml of working solution, add 27,300 U of collagenase.
Suspension buffer: To make 1 litre, add 750ml dH2O, 4g NaCl, 0.4g KCl, 0.13g MgCl2 × 6 H2O, 0.15g KH2PO4, 0.1 g Na2SO4, 7.2g HEPES, 6.9g TES, and 6.5 g Tricine while stirring. After dissolving, adjust pH to 7.6. Furthermore, add I g glucose, 30 g BSA, and 5 mg phenol red and sterilize by filtration. Keep at 4°C.
Pentobarbital solution: To get 1 ml, weigh 50mg of pentobarbital and complete to 1 ml with dH2O.
Heparin solution: To make 2.5ml, weigh 1 mg heparin sodium salt and complete to 2.5ml with 0.9% NaCl.
MEM culture medium with Hank's salts, L-gluthamine, and nonessential amino acids: To prepare 1 litre, add the specified volume of powdered MEM, 320mg NaHCO3, and 750 ml dH2O while stirring, complete to 1 litre, adjust pH to 7.6, and aliquot. Keep at 4°C. Before use, weigh 0.5µg/ml insulin from bovine pancreas and 50µg/ml gentamicin and add to medium.
Hepatocytes may be isolated from untreated young laboratory rats by the in situ perfusion method with collagenase buffer following the modified methods by Berry and Friend (1969) and Seglen (1976). Note: It needs a special authorisation and large experience to perform manipulations with laboratory animals. Therefore, the method described in the following is only a summary.
3. Normal Human Bronchial Epithelial Cells (NHBEC)
NHBEC may be isolated from explants from the bronchial airways of patients after lob- or pneumectomia (i.e., the resection of one of the lobes or of the lung at a hemithorax because of bronchial carcinoma) following the method of Lechner and LaVeck (1989) modified by Stock (2002).
Epith-o-ser culture medium with supplements (serum free): Thaw the frozen supplements delivered and add to 500 ml of basal medium. Prepare the medium before use.
L-15 medium: To make 1 litre, dissolve the specified volume of powdered L-15 in 750ml dH2O and complete to 1 litre with dH2O.
Coating solution: To make 100 ml solution, dissolve 1 mg fibronectine, 3 mg collagen R, and 1 mg BSA in 90 ml L-15 medium while stirring. Complete to 100ml with L-15 and filtrate. Keep at 4°C. Trypsin-EDTA solution: Aliquot the delivered solution (0.05% trypsin/0.02% EDTA in Ca2+- and Mg2+-free PBS).
Viability Test by Trypan Blue Method
Dilute 1 ml of cell suspension with 9ml culture medium or buffer, mix gently, extract 1 ml of this mixture, and add 1 ml trypan blue solution. Identify living (pale) and nonliving (blue) cells.
B. Micronucleus Assay
Peripheral lymphocytes are one of the most applied cell types for the identification of micronuclei in human tissue. Therefore, the method of the cytokineseblock micronucleus assay with human lymphocytes is described (Fenech, 2000).
RPM11640 cell culture medium: To prepare 1 litre of cell culture medium, add the specified volume of powdered RPMI 1640 as described in the catalogue to 750 ml dH2O while stirring. Complete to 890 ml with dH2O, adjust pH to 7.4, and store at 4°C. Before use, add 100 ml of FCS and 10 ml of penicilline-streptomycine and proof the pH to be 7.4.
Fixative: To make 100ml, add 75 ml of methanol and 25 ml of glacial acetic acid and keep at 4°C.
Staining solution: To make 100ml of stock solution (0.24 mM), take 100 µl acridine orange and complete to 100ml with dH2O. Store at 4°C. To make 100ml of working solution, add 1 ml of stock solution and 15 ml of Soerensen buffer.
Soerensen buffer: 60 mM Na2HPO4: 60 mM KH2PO4 = 1:1, pH 6.8.
Solution 1: To make 1 litre, weigh 11.876 g of Na2HPO4 and complete to 1 litre with dH2O.
Solution 2: To make 1 litre, weigh 9.078 g of KH2PO4 and complete to 1 litre with dH2O.
To get 100ml of buffer with pH of 6.8, add 50ml of Solution 1 and 50ml of Solution 2.
Perform the micronucleus test following the modified methods of Fenech and Morley (1985) and Sgura et al. (1997).
C. Alkaline Single Cell Gel Electrophoresis (Comet Assay)
NMA in aqueous solution: To make 100ml 1% agarose solution, add 1 g NMA to 100ml distilled water, heat carefully until the agarose is dissolved, and store at 4°C.
NMA in PBS: To make 100ml of a 0.6% solution, add 0.6 g NMA to 100ml PBS (without Ca2+ and Mg2+), heat carefully until agarose is dissolved completely, and store at 4°C.
LMA in PBS: To make 100ml of a 0.5% solution, add 0.5g LMA to 100ml PBS (without Ca2+ and Mg2+), heat carefully until agarose is dissolved completely, and store at 4°C.
Lysing solution: To make I litre, add 1.21 g Tris (10 mM), 146. 1 g NaCl (2.5M), and 37g Na-EDTA (100mM) to about 700ml dH2O while stirring. After dissolving, complete to 890 ml with dH2O, adjust pH to 10, and keep at room temperature. Immediately before use, add 1% Triton-X and 10% DMSO.
Electrophoresis buffer: To make 1 litre, add 0.37g Na-EDTA (1 mM) and 12g NaOH (300mM) to about 700 ml dH2O under stirring. After dissolving, complete to 1 litre with dH2O and adjust pH to 13. Prepare fresh.
Neutralisation buffer: Per litre, add 48g Tris to 750ml dH2O and complete to 1 litre with dH2O (400mM, pH 7.4).
Ethidium bromide solution: To get 10ml, dissolve 200µg EtBr in distilled water and complete to 10ml. Aliquot in 1-ml portions and keep at 4°C.
1. Preparation and Treatment Schedule of Cells
NHBEC (or Other Monolayer Cultures)
Short-term Treatment (1-2 hs)
Long-term Treatment (>2 hs)
2. Preparation of Cells from in vivo Experiments
Follow the procedures described earlier except for the treatment of cells.
Cells from Suspension Culture. Decant medium from cultured cells and resuspend cells gently in 85 ],tl melted LMA per slide.
Cells from Monolayer Culture
Note: For administration of LMA, do not exceed temperature of 37°C. Adjust cell number to 10,000 per slide. In human cell culture systems (lymphocytes and NHBEC), S9 mix as an external metabolising system should be involved (described in Section III,D) in parallel experiments. Do not treat cells longer than 3h with S9 mix.
3. Slide Preparation
The slide preparation follows the basic method of Singh et al. (1988). All steps following step 3 are performed in a dark room under yellow light.
D. Further Procedures
1. Preparation of S9 Mix from S9 Fraction
Most of the cell culture systems, apart from hepatocytes, should be exposed to test substance both in the presence and in the absence of an external metabolising system, i.e., the postmitochondrial supernatant of the rat hepatocyte preparation of Aroclor 1254 pretreated male rats ($9 mix) as described by Czygan et al. (1973), Ames et al. (1975), and Natarajan et al. (1976), to consider the effectivity of test substance in dependency on its metabolisation. Usually, the final concentration of $9 mix ranges from 1 to 10%, depending on the classification of test substance.
2. Preparation of S9 Fraction
3. Preparation of S9 Mix
Stock phosphate solution: (a) Dissolve 4.804 g KH2PO4 in dH2O and complete to 100 ml. (b) Dissolve 6.286 g Na2HPO4 in dH2O and complete to 100ml. Mix 19.6 ml of solution a with 80.4 ml of solution b, adjust pH to 7.4, and sterilize by heat (20min at 121°C in autoclave). Store at 4°C.
MgCl2: Dissolve 406.51 mg MgCl2 × 6 H2O in dH2O and complete to 50 ml, aliquot, and sterilize by microfiltration. Store at 4°C.
KCl: Dissolve 615mg KCl in dH2O and complete to 50ml and sterilize by microfiltration. Store at 4°C.
NADP: Dissolve 726 mg NADP in distilled water and complete to 20ml and sterilize by microfiltration. Aliquot and store frozen at -30°C.
Glucose-6-phosphate: Dissolve 304 mg glucose-6- phosphate in dH2O and complete to 20ml, aliquot, and sterilize by microfiltration. Store at -30°C.
In each experiment, in addition to the test concentrations, a negative control (culture medium) and a positive control should be included for validation of results [micronucleus test: 0.5M MMC, following Surral6s and Natarajan (1997), comet assay: 300µM H2O2]. If a test substance has to be dissolved or suspended in any vehicle, a vehicle control has to be included too.
It is important to determine the concentration range as well as the treatment period in prestudies for every test substance and for various cell systems. The number of different test concentrations should be three or more. For more detailed description of test parameters, see Tice et al. (2000).
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