CHAPTER 7 Detection of DNA Damage in Drosophila and Mouse ALOK DHAWAN*, MAHIMA BAJPAYEE AND DEVENDRA PARMAR Developmental Toxicology Division, Indian Institute of Toxicology Research (Formerly – Industrial Toxicology Research Centre), P.O. Box – 80, M.G. Marg, Lucknow – 226 001, India The single-cell gel electrophoresis (SCGE)/Comet assay has, since its incep- tion, been widely used for the simple, sensitive and rapid determination of DNA damage and repair, quantitatively as well as qualitatively in individual cell populations. 1 Comet is the perfect acronym for credible observation and measurement of exposure to toxicants. The assay combines the simplicity of biochemical techniques for detecting DNA single-strand breaks with the sin- gle-cell approach of cytogenetic assays. The advantages of the assay include its need for small numbers of cells per sample (o10 000), collection of data at the level of the individual cell, allowing for robust statistical analyses, its sensitivity for detecting quantitative and qualitative DNA damage. The assay has versatility in detecting DNA single- and double-strand breaks, oxidative DNA damage, crosslinks as well as apoptosis and necrosis in proliferating or nonproliferating cells, and has thus gained popularity as a test for genetic toxicology. Single cells obtained from various organisms ranging from simple bacteria (prokaryotes) to complex humans (eukaryotes) have been used to monitor in vitro or in vivo genotoxicity of chemicals. 2 It has also been used Issues in Toxicology No 5 The Comet Assay in Toxicology Edited by Alok Dhawan and Diana Anderson r Royal Society of Chemistry 2009 Published by the Royal Society of Chemistry, www.rsc.org * Corresponding author 151 Downloaded by North Carolina State University on 15 October 2012 Published on 27 August 2009 on http://pubs.rsc.org | doi:10.1039/9781847559746-00151
Transcript
CHAPTER 7
Detection of DNA Damagein Drosophila and Mouse
ALOK DHAWAN*, MAHIMA BAJPAYEE ANDDEVENDRA PARMAR
Developmental Toxicology Division, Indian Institute of Toxicology Research(Formerly – Industrial Toxicology Research Centre), P.O. Box – 80, M.G.Marg, Lucknow – 226 001, India
The single-cell gel electrophoresis (SCGE)/Comet assay has, since its incep-tion, been widely used for the simple, sensitive and rapid determination ofDNA damage and repair, quantitatively as well as qualitatively in individualcell populations.1 Comet is the perfect acronym for credible observation andmeasurement of exposure to toxicants. The assay combines the simplicity ofbiochemical techniques for detecting DNA single-strand breaks with the sin-gle-cell approach of cytogenetic assays. The advantages of the assay includeits need for small numbers of cells per sample (o10 000), collection of dataat the level of the individual cell, allowing for robust statistical analyses, itssensitivity for detecting quantitative and qualitative DNA damage. The assayhas versatility in detecting DNA single- and double-strand breaks, oxidativeDNA damage, crosslinks as well as apoptosis and necrosis in proliferating ornonproliferating cells, and has thus gained popularity as a test for genetictoxicology. Single cells obtained from various organisms ranging from simplebacteria (prokaryotes) to complex humans (eukaryotes) have been used tomonitor in vitro or in vivo genotoxicity of chemicals.2 It has also been used
Issues in Toxicology No 5
The Comet Assay in Toxicology
Edited by Alok Dhawan and Diana Andersonr Royal Society of Chemistry 2009
Published by the Royal Society of Chemistry, www.rsc.org
for ecogenotoxicology studies for assessing the genotoxicity of environmentalconditions on the sentinel species.3
In this chapter, the general protocol for the assessment of DNA damage inany cell type as well as protocols utilised in mouse and Drosophila melanogasterfor evaluation of in vivo genotoxicity are discussed.
7.1 General Protocol for the Assessment of DNA
Damage Using the Alkaline Comet Assay
The Comet assay may be used in any cell type that can be obtained as a single-cell suspension. The cells may be of animal, plant or human origin. The whiteblood cells are the most frequently used cell type for the Comet assay in humanbiomonitoring studies,4,5 however, other cells have also been used, e.g. buccalcells,6 nasal,7 sperm,8–10 epithelial11 as well as placental cells.12 The Cometassay has also been used for detecting the genotoxicity in plant models13,14 withcells from leaves,15 stems and roots.16 From animals, blood lymphocytes, bonemarrow cells, and cells from organ/tissues such as liver, brain, and spleen havealso been used.17–19
Guidelines for conducting the assay have been formulated and recommen-dations have been published.20,21 Detailed protocols for performing the assay indifferent samples and for different types of DNA damage are also available onthe Comet assay website (www.cometassayindia.org). The general protocol forconducting the Comet assay in different models has been depicted in Figure 7.1.
mm, with 19mm frosted end), coverslips (No. 1, 24� 60mm), frozen ice packs,microcentrifuge tubes, micropipettors and tips, Coplin jars (opaque), micro-scope slide tray (aluminium).
7.1.2 Preparation of Reagents
1. PBS (Ca21, Mg21 free): 1 L packet of Dulbecco’s PBS is added to990mL distilled water (dH2O), pH adjusted to 7.4, and volume made upto 1000mL. Stored at room temperature.
2. Low melting point agarose: Prepare 1% (500mg per 50mL PBS) and0.5% LMPA (250mg per 50mL PBS). Microwave or heat until nearboiling and the agarose dissolves. Aliquot 5mL samples into scintilla-tion vials (or other suitable containers) and refrigerate until needed.
153Detection of DNA Damage in Drosophila and Mouse
When required, briefly melt agarose (60–701C) in a microwave or byanother appropriate method. Place LMPA vial in a 37 1C dry/waterbath to cool and stabilise the temperature. Do not keep heating theagarose or the concentration will change.
3. Normal melting agarose NMA (1.0%): 500mg per 50mL in Milli Qwater. Microwave or heat until near boiling and the agarose dissolves.Maintain temperature B 60 1C in a dry bath for use.
4. Lysing solution: Ingredients per 1000mL:2.5MNaCl 146.1 g100mM EDTA 37.2 g10mM Trizma base 1.2 g
Add all the above ingredients to about 700mL dH2O with B8 g NaOHand allow the mixture to dissolve on a stirrer. pH is adjusted to 10 usingconcentrated HCl or NaOH. q.s. to 1000mL with dH2O and stored thestock at room temperature. NaOH is used for dissolving EDTA.Final lysing solution is prepared fresh before each experiment. Add 10%DMSO (in the case of haeme-containing cells) and/or 1% Triton X100to the stock lysing solution and then refrigerate for at least 30min priorto slide addition.
NOTE: The purpose of the DMSO in the lysing solution is to scavengeradicals generated by the iron released from haemoglobin when blood oranimal tissues are used. It is not needed for other situations.
Stored at room temperature, and fresh stock solutions of NaOH andEDTA can be prepared every B2 weeks. (Dissolve the EDTA with helpof NaOH pellets or concentrated NaOH.)For use, 1X Buffer is made fresh before each electrophoresis run.
Mix 30mL NaOH and 5mL EDTA stock solutions and makeup to 1000mL with chilled dH2O. The total volume depends onthe gel box capacity. Prior to use, the pH of the buffer has to be ensuredto be 413.
6. Neutralisation buffer (0.4M Tris): 48.5 g is added to B800mL dH2O,adjusted pH to 7.5 with concentrated (410M) HCl made up to 1000mLwith dH2O, store at room temperature. Use chilled.
7. Staining solution: ethidium bromide (EtBr; 10X Stock – 200 mg/mL): add10mg in 50mL dH2O, and store at room temperature. For use, 1Xsolution is prepared with 1mL stock and 9mL dH2O.
CAUTION: Handle EtBr with adequate precautions as it is a knowncarcinogen.
7.1.3 Preparation of Agarose-Coated (Base) Slides for the
Comet Assay
1. The conventional microscope slides (with 19 mm frosted end) are dippedin methanol and burnt over a blue flame to remove the machine oil anddust. (If precleaned slides are available then this step can be omitted).
2. While NMA (1%) is hot (601C), the slides are dipped up to one-third thefrosted area and gently removed. The underside of the slide is wiped toremove agarose and the slides laid on a flat surface to dry. Do not touchthe slide to the side of the beaker or agarose runs off.
3. The slides may be air dried or warmed at 50 1C for quicker drying, andstored at room temperature until needed, avoiding high-humidity con-ditions. Generally, slides are prepared a day before use.
NOTE: Slides should be labelled on the agarose side before storage.
7.1.4 Preparation of Microgel Slides for the Comet Assay
1. Cells of interest (whole blood, lymphocytes, cells from various tissues)are diluted with PBS and equal volumes of diluted cells (100 mL) and 1%LMPA (100 mL) are mixed. 80 mL of this mixture are placed onto twoduplicate slides. Alternatively, to each of the coated slides 75 mL ofLMPA (0.5%; at 37 1C) mixed with B10 000 cells in B5–10 mL (do notuse more than 10 mL) are added.
2. Coverslips are placed on the slides to evenly spread the gel.3. The slides are placed on a slide tray resting on ice packs until the agarose
layer hardens (B5 to 10min).4. Coverslips are gently removed and a third agarose layer (80 mL LMPA;
0.5%) is added to the slide.
Note: The final concentration of LMPA in the second and third layersshould be the same to prevent uneven migration of DNA in the two layers.
5. Replace the coverslip to evenly spread the gel and return to the slide trayuntil the agarose layer hardens (B5 to 10min).
6. The coverslips are finally removed and the slides carefully put intoCoplin jars containing chilled, freshly prepared final lysing solution.
7. The slides are protected from light and refrigerated for a minimum of1 h. The slides may be stored for at least 4 weeks in cold lysing solutionwithout affecting the results. (The Lysing time may depend on the celltype and should be standardised.)
NOTE: The amounts indicated are based on using No. 1, 24� 60mmcoverslips. Proportional volumes can be used for coverslips differing insize. If the gels are not sticking to the slides properly, avoiding humidityand/or increasing the concentration of NMA agarose in the lower layer to1.5% should eliminate the problem. Steps 4 to 6 should be performedunder dim yellow lights to prevent additional DNA damage.
155Detection of DNA Damage in Drosophila and Mouse
The procedure described here is for electrophoresis under pH413 alkalineconditions.
1. After lysis at B4 1C, slides are gently removed from the lysing solutionand placed side by side in the horizontal gel box near one end, slidingthem as close together as possible.
2. The buffer reservoirs are filled with freshly made, chilled 1X electro-phoresis buffer (pH413) until the liquid level completely covers theslides (avoid bubbles over the agarose).
3. Let slides sit in the alkaline buffer for 20min to allow for unwinding ofthe DNA and the expression of alkali-labile damage.
NOTE: The longer the exposure to alkali, the greater the expression ofalkali-labile damage.
4. The power supply is turned on to 24V (B0.7V/cm) and the currentadjusted to 300 milliamperes by raising or lowering the buffer level.Depending on the purpose of the study and on the extent of migration incontrol samples, electrophoresis is carried out for 10 to 40min.
NOTE: The goal is to obtain migration among the control cells withoutit being excessive. The optimal electrophoresis duration differs fordifferent cell types. If crosslinking is one of the endpoints being assessedthen having controls with about 25% migrated DNA is useful. Alower voltage, amperage and a longer electrophoresis time may allowfor increased sensitivity. Different gel boxes will require differentvoltage settings to correct for the distance between the anode and thecathode. The electrophoresis should be carried out between 0.7–1V/cm.
5. After electrophoresis, the slides are lifted from the buffer and placed on adrainage tray. Neutralising buffer (pH7.5) is added dropwise to coat theslides and allowed to sit for at least 5 min. The slides are drained and thisstep is repeated two more times.
6. These slides may be either stained and scored immediately or dried forlater processing.
a. Slides are stained with 80 mL 1X EtBr for 5min. Then, the slides aredipped in chilled distilled water to remove excess stain, a coverslip isplaced over it and the slides are stored in a humidified slide box untilscoring.
b. For drying, the slides are kept in cold 100% ethanol/methanol fordehydration for 20min. The slides are air dried and then placed in anoven at 50 1C for 30min. The slides are then stored in a dry box. Whenconvenient, the slides are rehydrated with chilled distilled water for30min and stained with EtBr as above and covered with a fresh
coverslip. For archival purposes, the slides after scoring, are destainedwith 100% alcohol, dried and stored dry.
NOTE: Perform steps 1 through 6 under yellow light; the premise is thatnormal lighting will cause DNA damage.
7.1.6 Evaluation of DNA Damage
1. EtBr-stained DNA damage is visualised using a 40� objective on afluorescence microscope.
2. Although any image-analysis system may be suitable for the quantitationof SCGE data, we use Komet 5 image-analysis software developed byKinetic Imaging, Ltd. (Liverpool, UK). The software is linked to a CCDcamera to assess the quantitative and qualitative extent of DNA damagein the cells by measuring the length of DNA migration and the percentageof migrated DNA. Generally, 50 to 100 randomly selected cells are ana-lysed per sample. Finally, the program calculates and automaticallygenerates the values for tail (%) DNA, tail length, and tail moment. Thetail moment is defined as the distance between the centre of mass of thetail and the centre of mass of the head, in micrometres, multiplied by thepercentage of DNA in the tail and is considered to be the most sensitive asboth the quality and quantity of DNA damage are taken into account.
3. The amount of migration per cell, the number of cells with increasedmigration, the extent of migration among damaged cells is thencompared.
7.2 The Alkaline Comet Assay in Multiple
Organs of Mouse
The Comet assay is now a well-established supportive assay to the standardbattery of genotoxicity tests and recommended as an in vivo test in the secondstage of genotoxicity testing.22 It can be used to investigate the potentialmechanisms of tumorigenic responses and to evaluate genotoxicity of chemicalsthat are positive in other in vivo mutagenicity tests. Guidelines and recom-mendations for performing the in vivo assay have been developed.21,23 The mostimportant advantage provided by the Comet assay for assessing the geno-toxicity in vivo is that the DNA damage can be measured in cells of any organ,regardless of the extent of mitotic activity. Cytogenetic techniques like themicronucleus assay, chromosomal aberration test and the sister chromatidexchange assay, require a proliferating cell population for assessing genotoxi-city in cells, e.g. cells of the haematopoietic system. However, several chemicalspass the blood–organ barrier, reach the organs and elicit their toxic responseincluding genotoxicity. Hence, genotoxicity in organs cannot be assessed usingconventional cytogenetic techniques unless the cells are made to undergo
157Detection of DNA Damage in Drosophila and Mouse
mitosis. Therefore, Sasaki et al.24,25 devised a method to assess multiorgangenotoxicity in the mouse using the alkaline Comet assay using a homo-genisation technique to detect the genotoxicity of chemicals in vivo in theirtarget organs. Using this method, each organ was minced, suspended in chilledhomogenising buffer containing NaCl and Na2EDTA, gently homogenisedusing a Potter-type homogeniser set in ice, and the centrifuged nuclei were usedfor the alkaline Comet assay.24 Later, Sasaki et al. also compiled the data ofgenotoxicity of 208 carcinogenic chemicals from the IARC database using thealkaline Comet assay.17
The assay showed a high positive response ratio for rodent genotoxic car-cinogens and a high negative response ratio for rodent genotoxic noncarcino-gens.17 The findings suggest that the alkaline Comet assay can be usefully usedto evaluate the in vivo genotoxicity of chemicals in multiple organs, providingfor a good assessment of potential carcinogenicity.17 The assay has been used todetermine the threshold dose at which it has beneficial or toxic effects.26
Ueno et al. carried out DNA damage and repair studies in multiple organs ofwhole-body X-irradiated mice that suggested differences in the radiosensitivityof nuclear DNA and DNA-repair capacity among organs.27 A comparativeinvestigation of species differences in genotoxicity in multiple organs of miceand rats using the Comet assay was conducted by Sekihashi et al.28 sincesensitivity to xenobiotics is different for different species and species differencesin carcinogenicity for mice and/or rats is known.Here, we describe the methodology followed in our laboratory for per-
forming the in vivo Comet assay in multiple organs of mouse (Figure 7.2).19
7.2.1 Chemicals and Materials
Chemicals and materials for the Comet assay were as described earlier in 7.1.However, Heparin Hank’s balanced salt solution (HBSS), Histopaque-1077,
and RPMI-1640 medium are also required in studies involving mice.
7.2.2 Methodology
7.2.2.1 Animals
Male Swiss albino mice (o6-weeks old, 20� 2 g) obtained from the breedingcolony at our institute were raised on a commercial pellet diet and waterad libitum. Animals were cared for in accordance with the policy laid down bythe Animal Ethics Committee of our Institute.
7.2.2.2 Treatment
Experiments were planned according to the Comet assay guidelines.20,21 4 or 5mice were included in each treatment group and caged separately. They weretreated intraperitoneally for 5 consecutive days with the test sample (e.g.cypermethrin19).
7.2.2.3 Sacrifice and Collection of Tissue Samples
Blood was drawn from the orbital sinus and 20–50 mL collected into hepar-inised Eppendorffs tubes.Animals were sacrificed by cervical dislocation. Organs (brain, liver, kidney and
spleen) were dissected out and collected immediately in cold HBSS medium.
Liver
20 µl Blood +1 ml RPMI layered over 100 µl Histopaque-1077
Both the femurs were dissected out and cleaned thoroughly to remove musclesand other tissue. Bone marrow cells were flushed in 1 mL PBS using a syringe.
7.2.2.4 Single-Cell Preparation
i) Blood lymphocytes: the lymphocytes were isolated from whole bloodusing Histopaque-1077 by the method of Boyum29 with slight mod-ifications. Briefly, 20 mL of blood was added to 1mL RPMI-1640 andlayered over 100 mL Histopaque. This was centrifuged at 500�g for3min. The interface of medium/Histopaque containing the lymphocyteswas taken and added to 1 mL medium (RPMI-1640). It was then cen-trifuged at 500�g for 3 min to pellet the lymphocytes, which wereresuspended in PBS for the Comet assay.
ii) Preparation of a single-cell suspension from organs was done accordingto the method of refs. 18 and 20. Briefly,o0.2 g of each organ was placedin 1 mL of freshly prepared chilled mincing solution (Hank’s balancedsalt solution, with 20mM EDTA and 10% DMSO) in a Petri dish andchopped into pieces with a pair of scissors. The pieces were allowed tosettle and the supernatant containing the single cells was taken.18 Cellcounting was done and a cell suspension of B20 000 cells in 100 mL PBSwas used for the studies.
7.2.2.5 Cell Count and Cell Viability Assay
Cells from organs and tissues were counted using a haemocytometer anddiluted with PBS to achieve a concentration of 0.2�106 cells/ml. The viability ofcells isolated from liver, spleen, kidney and brain was checked by 5,6-carboxy-fluorescein dye,30 while trypan blue was used for the blood and bone marrowcells.31
7.2.2.6 Single-Cell Gel Electrophoresis/Comet Assay
i) Preparation of slides and lysis: The base slides were prepared with 1%NMA using conventional end frosted slides as discussed earlier.On this base layer, 80 mL of sample (containing 100 mL cell suspensionmixed with 100 mL of 1% LMPA) was added to form the second layer.A coverslip was placed gently to evenly spread the cells in the agarose.After the gel solidified, a third layer of 0.5% LMPA (90 mL) was addedonto the slide and placed over ice for 10min. Finally, the coverslipswere removed and the slides immersed in freshly prepared chilled lysingsolution containing 2.5M NaCl, 100mM EDTA, 10mM Tris (pH10)with 10% DMSO and 1% Triton X-100 being added just before use.The slides remained in the lysing solution overnight at 4 1C.
ii) Electrophoresis: Electrophoresis was carried out according to themethod of Singh et al.32 as described earlier. The slides were placed in a
horizontal gel electrophoresis tank (Life Technologies, Gaithersburg,USA) side-by-side and avoiding spaces, with the agarose ends nearest tothe anode. Fresh and chilled electrophoresis buffer (1mM Na2EDTAand 300 mM NaOH, pH413) was poured into the tank to cover theslides. The slides were left in this solution for 25min to allow DNAunwinding and expression of alkali-labile sites as DNA-strand breaks.Electrophoresis was conducted at 24V (0.7V/cm) and a current of 330mA using a power supply (Electra Comet III from Techno SourceIndia Pvt. Ltd., Mumbai, India) for 30min at 4 1C. All steps wereperformed under dimmed light to avoid additional DNA damage due tostray light.
iii) Neutralisation and staining: After electrophoresis, the slides were drainedand placed horizontally in a tray. Tris buffer (0.4 M; pH7.5) was addeddropwise and left for 5min to neutralise excess alkali. The procedure wasrepeated thrice. The slides were stained with 75mL EtBr (20mg/mL) for5min and dipped in chilled distilled water to wash off excess EtBr, and acoverslip placed over them. Slides were placed in a dark humidified slidebox to prevent drying of the gel. The slides were scored within 24h.
iv) Scoring of slides: Slides were scored using an image-analysis system(Kinetic Imaging, Liverpool, UK) attached to a fluorescence micro-scope (Leica, Germany) equipped with appropriate filters (N2.1, exci-tation wavelength of 515–560 nm and emission wavelength of 590 nm).The microscope was connected to a computer through a charge-coupleddevice (CCD) camera to transport images to software (Komet 3.1) foranalysis. The final magnification was 400�. The comet parametersrecorded were Olive tail moment (OTM, arbitrary units), tail DNA (%)and tail length (TL; mm). Images from 100 cells (50 from each replicateslide) were analysed.
7.2.2.7 Statistics
Homogeneity of variance and normality assumption of data were tested and wasfound to be normally distributed. The mean values of OTM, tail % DNA, andTL at each concentration of the test sample were compared with the negativecontrol using one-way ANOVA18 at Po0.05 level of statistical significance.
7.3 The Alkaline Comet Assay in Drosophilamelanogaster
Drosophila melanogaster, or the fruit fly, has been intensely studied for almost 100years. It is a complex multicellular organism with many aspects of its developmentand behaviour parallel to those in human beings. Unique genetic and moleculartools have evolved for analysis of gene function in this organism. These advan-tages have allowed the use of Drosophila for the understanding of fundamentalbiological processes and provide unique insights in the genomic era. Drosophilahas hence found extensive use as a model organism in various fields including
161Detection of DNA Damage in Drosophila and Mouse
toxicology testing.33–38 Concerns have been raised about the ethics and use ofanimals for toxicology research and testing, and emphasis now is given to the useof alternatives to mammals in research as well as education. The European Centrefor the Validation of Alternative Methods (ECVAM) promotes scientific andregulatory acceptance of alternative methods for reducing, refining or replacingthe use of laboratory animals39,40 and recommends D. melanogaster as an alter-native to animal models for testing and research.Here, we describe the usefulness of D. melanogaster as an in vivo model for
assessment of genotoxicity using the alkaline Comet assay (Figure 7.3).
7.3.1 Chemicals and Materials
All chemicals and materials for the Comet assay were as described earlier in 7.1.However, Poel’s salt solution, sodium phosphate buffer, and collagenase are
also required in studies involving D. melanogaster.
7.3.2 Methodology
7.3.2.1 Drosophila melanogaster
The wild-type (Oregon R+) fly and larvae of D. melanogaster were cultured at24� 1 1C and grown on a standard diet of agar, corn meal, brown sugar andyeast.Freshly emerged first or third instar larvae (22� 2 h) were then used for the
genotoxicity studies.The larvae were fed on a diet of standard Drosophila food containing dif-
ferent concentrations of genotoxicants, e.g. cypermethrin41 or industrial lea-chates,42,43 and allowed to grow on it. Larvae grown only on standardDrosophila food constitute the negative control, while those fed ethyl metha-nesulfonate, a known mutagen44 constitute the positive control.
7.3.2.2 Preparation of Cell Suspension
10–50 larvae are used for preparing cell suspensions for each concentration. At96� 2 h, the larvae were removed from the food and washed with 50mMsodium phosphate buffer. Brain ganglia and the anterior region of the midgutare dissected and explanted in Poels’ salt solution (PSS).45 A single-cell sus-pension of the issues was then prepared by the modified method of Howell andTaylor.46 PSS was replaced with collagenase (0.5mg/mL in PBS, pH 7.4) andcells incubated for 15min at 24 1C. The cells were then passed through nylonmesh (60mm). Collagenase was removed by washing the cell suspension threetimes with PBS. The cells were finally suspended in 80mL of PBS.
The Comet assay technique still requires some modification and stand-ardisation under different experimental conditions and/or using differentexperimental materials. The modifications and their reasons are discussedbelow:
i) Slide preparation: Slides were prepared in duplicate for each con-centration as discussed earlier in the chapter. The base slides were pre-pared with NMA (1%) as discussed earlier.
However, owing to the difference in the size of the cells, modification in theconcentration of LMPA was made. Generally, 1% LMPA is mixed with thecells in equal volumes (final concentration 0.5%) and is recommended for use inthe second layer.20,44 Since the midgut and brain ganglia cells of Drosophila aresmaller than mammalian cells, for our studies equal volumes of 1.5% LMPA(0.75% final concentration) and cell suspension were mixed. Similarly, the thirdlayer consisted of LMPA (0.75%).
ii) Lysing: A major modification was made in the composition of thelysing solution as compared with that used by Bilbao et al.44 This wasremoval of DMSO from the final lysing solution. DMSO is recom-mended at 10% and is usually added to scavenge radicals generated bythe iron released from haemoglobin.32 However, no such hemegroups are present in Drosophila. No scorable cells could be detectedwhen slides were placed in lysing solution containing DMSO as usedconventionally. Also, an earlier study had shown that a dietary con-centration of over 0.3% DMSO was cytotoxic to D. melanogaster.47
Thus, in the final lysing solution DMSO was not added. The slides werefinally immersed in freshly prepared chilled lysing solution (2.5MNaCl, 100mM EDTA, 10mM Tris pH10.0 and 1% Triton X-100,pH10) for 2 h.
iii) Electrophoresis: After lysis, the slides were placed in a horizontal gelelectrophoresis tank (Life Technologies, Gaithersburg, MD) filled withfresh, chilled electrophoresis solution (1mM Na2EDTA and 300mMNaOH, pH413). Although Bilbao et al.44 in their study used 20minunwinding and electrophoresis of neuroblast cells of Drosophila, in ourlaboratory, no scorable cells could be observed when the time wasmaintained at 20min. The experimental conditions were optimised inour laboratory and the times of unwinding and electrophoresis werereduced to 10 and 15min, respectively, resulting in an improvement inperformance of the assay. Electrophoresis was conducted at 0.7V/cmand 300mA at 4 1C using a power supply.
iv) Neutralisation and staining of slides: Tris buffer (0.4M Tris pH 7.5)was added dropwise to neutralise excess alkali and procedurerepeated thrice. Slides were then stained with EtBr (20mg/mL, 75 mL perslide) for 10min in the dark. They were dipped in chilled distilledwater to remove excess stain and subsequently coverslips were placed on
them. The prepared slides were kept in a humidified slide box untilscoring.
v) Scoring: The slides were scored as discussed earlier using a fluorescentmicroscope attached to a CCD camera. 25 cells per slide were randomlycaptured, avoiding the cells present in the edges of the gel and super-imposed comets.
Each experiment was performed in triplicate with 10–50 larvae.
7.3.2.4 Statistics
Prior to analysis, homogeneity of variance and normality assumptions con-cerning the data were tested. The mean values of the Comet parameters werecompared using the Student’s t-test.
7.4 Conclusions
The Comet assay has gained wide acceptance in genotoxicity testing due to itssimplicity, and sensitivity. The added advantage of being simple and able todetect different types of DNA damage in any cell, regardless of its proliferatingstatus makes the assay more versatile. This has resulted in widespread pro-gression of this technique in many areas, e.g. environmental monitoring,3
human monitoring,5,48–50 genetic toxicology.51 The assay has been accepted byinternational guidelines as an in vivo test52 and guidelines as well as recom-mendations have been published.20,21,23
In this chapter, the use of the Comet assay in assessing DNA damage in twoin vivo models, i.e. an animal model, mouse, and an alternate to the animalmodel, Drosophila, have been discussed.The Comet assay in rodents is an important test model for genotoxicity
studies, since it provides an insight into the genotoxicity and its underlyingmechanisms of human carcinogens (since many rodent carcinogens are knownto be human carcinogens). The mouse organs exhibiting increased levels ofDNA damage may not be the target organs for carcinogenicity. Therefore, forthe prediction of carcinogenicity of a chemical, organ-specific genotoxicity wasnecessary but not sufficient.17 The Comet assay can also be used as an in vivotest for assessing DNA damage for those compounds which have poor systemicbioavailability. Multiple organs of mouse/rat including brain, blood, kidney,lungs, liver, bone marrow have been utilised for the comprehensive under-standing of the systemic genotoxicity of chemicals.17,18,28,53,54
Also, since the scientific world is moving towards a reduction in theuse of animals in toxicity testing, alternatives to animal models becomeimportant. One such organism is the fruit fly Drosophila melanogaster. Thismodel was earlier used mostly for germ cell mutagenicity studies, however,recently, it has gained importance in studying somatic cell genotoxicity ofchemicals.41–44
165Detection of DNA Damage in Drosophila and Mouse
Although the Comet assay is a useful technique, the variability in results fromdifferent laboratories, interpretation of the results and lack of validated studiesare some of its disadvantages. Care should be taken during preparation of thesingle-cell suspension so that viable cells are obtained for analysis. Also, via-bility of the cells should be checked before proceeding with the Comet assay sothat the DNA damage observed is due to the genotoxicity of the compound andnot due to DNA fragmentation of damaged/dead cells. Each step of the assayshould be conducted under the proper conditions (pH, temperature) to reduceambiguous results. The International Workgroup on Genotoxicity Testing(IWGT) has discussed study design and data analysis in the Comet assay andemphasis was given to the alkaline version (pH413) of the in vivo Comet assayand recommendations were made for a standardised protocol, which would beacceptable to international agencies.23 Scoring manually or with the help ofautomated software are both allowed, however, user bias can be reduced byscoring random cells. The statistical analysis in the Comet assay takes intoaccount the study design and has been well reviewed.55,56 Both univariate andmultivariate analyses can be conducted on the results obtained in the assay.The Comet assay in in vivo models such as mice and Drosophila allows the
assessment of genotoxicity of chemicals that can mimic the responses inhumans. These models thus provide an understanding of the mechanismof genotoxicity57 as well as the response of biological systems to thesechemicals.
Acknowledgements
The authors wish to thank the Council of Scientific and Industrial Research(CSIR), New Delhi, India for funding through the Networked Projects(CMM0018 and NWP34) as well as the support from UK-India Education andResearch Initiative (UKIERI).
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