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Cell Biology International (2004) 28, 497–502 (Printed in Great Britain)
The comet assay differentiates efficiently and rapidly between genotoxins and cytotoxins in quiescent cells
P Daza*, J Torreblanca and F.J Moreno
Department of Cell Biology, Faculty of Biology, Avda. Reina Mercedes 6, 41012 Sevilla, Spain


Abstract

Our main aim was to establish the efficiency of the single cell electrophoresis technique for differentiating between drugs that bind DNA and those that do not. The alkaline comet assay was used to test the responses of human leukocytes (quiescent cells) to damage induced by reportedly genotoxic and reportedly cytotoxic agents. Incubation of G0 leukocytes for 1h with the genotoxic agents camptothecin and actinomycin C provoked DNA migration, observed as comet figures. On the other hand, when cells were treated with the cytotoxic agents cordycepin, fluorodeoxyuridine and puromycin, the leukocyte nuclei were indistinguishable from those of untreated cells. In addition, we have developed a rapid method using non-proliferating cells that requires neither culture nor lymphocyte isolation. This method promises to be useful as a rapid in vitro screening assay.


Keywords: Comet assay, Genotoxins, Cytotoxins, Quiescent cells.

*Corresponding author. Fax: +34-95-4610-261.


1 Introduction

Many techniques for detecting DNA damage have been used to identify potentially genotoxic substances. During recent years the comet assay or single cell gel electrophoresis technique has been established as a sensitive method for detecting DNA damage; it promises to be a rapid in vitro screening assay for in vivo genotoxins.

The comet assay was developed by Östling and Johanson (1984) for the direct visualization of DNA damage. In the alkaline version of the assay, as described by Singh et al. (1988), a dilute suspension of single cells in a thin agarose sandwich is lysed at pH 10 to release individual nuclei. These are incubated in an alkaline electrophoresis solution to facilitate DNA unwinding, after which DNA fragments from single nuclei are separated by a weak electric field. Depending on the amount of DNA damage, comets are observed after electrophoresis and staining with a fluorescent dye.

Although this technique is recognized as a good biomonitoring assay for classifying a wide range of compounds as clastogenic, the standard protocol seems unable to detect the damage caused by crosslinking agents (Hartmann and Speit, 1995); such agents retard DNA migration (Olive et al., 1992; Merck and Speit, 1999). Furthermore, it has been reported that the cellular damage caused by cytotoxic agents cannot be detected by single cell electrophoresis (Henderson et al., 1998).

The comet assay has been used during the last years for numerous in vivo and in vitro studies to monitor exposure to mutagens and carcinogens that induce DNA damage. In molecular epidemiological studies, DNA damage evaluated by the comet assay is utilized as a biomarker of exposure. The analysis of individual cells from storks and kites living in Doñana National Park indicated an increase in genotoxic damage, as shown by a significant increase in DNA migration in electrophoresed nuclei (Pastor et al., 2001a,b). More recently, other authors have observed an increase in the comet assay parameters of mice from the same National Park, indicating that the comet assay is an interesting and sensitive tool for studying environmental genotoxicity (Festa et al., 2003).

This technique has been applied to the monitoring of DNA damage in plant as well as animal cells, as in the studies of Navarrete et al., (1997) in Allium cepa and Menke et al., (2000) in Vicia faba cells. Navarrete et al., (1997) developed a fast comet assay for solid plant tissue cells. Menke et al., (2000) described a method based on a combination of comet assay and FISH to detect specific DNA lesions.

During the last decade, this assay has been adopted by investigators in many different research areas. Compared with other genotoxicity assays it offers numerous advantages: the ability to detect low levels of damage, the requirement for only small numbers of cells per sample, low costs, and ease of application. However, the single cell gel assay must undergo appropriate multilaboratory, international validation studies to confirm its reproducibility (Tice et al., 2000).

The aim of the present study was twofold: to confirm the ability of the comet assay to differentiate between genotoxins and cytotoxins, and to develop a method applicable to G0 human leukocytes, which do not require to be cultured. The data obtained support the view that this assay is a rapid method for screening these two types of cell-damaging agents.

2 Material and methods

2.1 Chemicals

The topoisomerase I inhibitor, camptothecin (CPT), the RNA synthesis inhibitors, actinomycin D (Ac D) and cordycepin (Cordy), the DNA synthesis inhibitor, fluorodeoxyuridine (FdU), and the protein synthesis inhibitor, puromycin (Puro), were purchased from Sigma Chemical Co. (St. Louis, MO, USA). CPT was dissolved in dimethyl sulfoxide (DMSO); the volume used for treatment never exceeded 1% of the total assay volume. All the other drugs were dissolved in distilled water.

2.2 Cell culture and treatments

Leukocytes were obtained from peripheral venous blood of a healthy male donor. Heparinized blood (500μl) was mixed with 4.5ml phosphate buffer saline (PBS) and aliquoted in 500μl vials. Cells were treated for 1h with the inhibitors during G0. The doses used for the different compounds were: (1) puromycin 0.5, 1 and 2mM; (2) actinomycin D 0.01, 0.02 and 0.04mM; (3) camptothecin 7.5, 15 and 30μM; (4) cordycepin 0.1, 0.2 and 0.4mM; (5) FdU 0.5, 1 and 2μM. After each of these treatments, the cells were washed with PBS and then immediately used for the comet assay under alkaline conditions.

2.3 DNA–comet assay (alkaline)

Regular slides were coated with a 1% solution of standard agarose in distilled water by immersing vertically for 2s and air-dried to solidify the agarose. Once the slides were dry and totally transparent, they were kept at 4°C and used up to 1 month after preparation. The blood cells were centrifuged and washed with PBS to remove the chemicals. We employed a modification of the protocol described by Singh et al. (1988) and Fairbairn et al. (1995). The cells (50–100μl) were suspended in a 0.7% low melting agarose solution in PBS and immediately pipetted on to the coated slides. A coverslip was added and the slides were incubated at 4°C for 10min. The coverslips were removed and a third (low melting point) agarose layer was added, together with new coverslips, and the incubation at 4°C for 10min was repeated. The coverslips were removed and the cells were lysed by incubating for 1h at 4°C in the dark in a lysis solution containing: 10mM Tris–HCl, 2.5M NaCl, 100mM Na2-EDTA, 0.25M NaOH, 1% (v/v) Triton X-100 and 10% (v/v) DMSO, pH 12. In order to unwind the DNA, the slides were incubated for 20min in electrophoresis buffer, which comprised 1mM Na2-EDTA and 300mM NaOH, pH 12.8. Electrophoresis was carried out at 1V/cm for 20min. After neutralization with 3×5min washes in 0.4M Tris–HCl pH 7.5, the cells were stained with 4′,6-diamidino-2-phenylindole (DAPI).

Cells were analysed using a Leica Q-win program. Two parameters were estimated for each comet: (1) integral fluorescence, which is proportional to the DNA content in the tail or in the head, and (2) tail length. Cells were categorized into ten different damage classes, from 1 to 10, in which class 1 were cells with DNA damage 10% (least damage), class 2=DNA damage between 10 and 20%, class 3=DNA damage between 20 and 30%, class 4=DNA damage between 30 and 40%, class 5=DNA damage between 40 and 50%, class 6=DNA damage between 50 and 60%, class 7=DNA damage between 60 and 70%, class 8=DNA damage between 70 and 80%, class 9=DNA damage between 80 and 90% and class 10 were cells in which the DNA damage level was higher than 90% (highest damage).

The tail moment was calculated by multiplying the percentage DNA damage by the tail length of the comet. All experiments were performed three times and for each experimental point at least 50 comets were measured. Student's t-test was used for statistical evaluation.

3 Results

The DNA comet assay results for cells treated with each of the chemicals are presented in Fig. 1. After treatment with puromycin, cordycepin or fluorodeoxyuridine (reportedly cytotoxins), most of the cells were distributed between classes 2 and 3, as were the control cells. However, cells treated with actinomycin D or camptothecin (genotoxins) presented a broader spectrum of damage and, as the histograms show, most of the cells were distributed in classes 8–10 at the highest doses (Fig. 1A–F). In Fig. 2 representative pictures for each of the treatments are provided.


Fig. 1

Histogram showing the frequency (%) of damaged cells for each treatment: (A) control, (B) puromycin, (C) cordycepin, (D) fluorodeoxyuridine, (E) actinomycin D, (F) camptothecin.


Fig. 2

Pictures of control and treated cells (A) Intact nuclei: control cells, as well as cells treated with Fluorodeoxyuridine, Cordycepin and Puromycin; (B) Damaged nuclei: Actinomycin D; (C) Damaged nuclei: Camptothecin.



Table 1 shows the average tail moments for all the treated and control cells. The data demonstrate that only those chemicals that directly affect DNA, the genotoxins, lead to average tail moments that are significantly different from control values (Student's t-test, p<0.001).


Table 1. Average tail moment values for the different inhibitors

Image

In Fig. 3, the tail moment values for the different inhibitors are grouped in intervals. The data refer only to the intermediate drug doses. Once again, it is clear that only the DNA binding drugs induced the formation of comets with significant tails. For cells treated with Puro, FdU and Cordy, the moments were always distributed in values 0–2, as were those for control cells. On the other hand, the moments were always grouped among values 4–10 after treatment with CPT or Ac D.



Full-size image (25K) - Opens new windowFull-size image (25K)
* Significant differences p<0.001 respect to control values.

In Fig. 3, the tail moment values for the different inhibitors are grouped in intervals. The data refer only to the intermediate drug doses. Once again, it is clear that only the DNA binding drugs induced the formation of comets with significant tails. For cells treated with Puro, FdU and Cordy, the moments were always distributed in values 0–2, as were those for control cells. On the other hand, the moments were always grouped among values 4–10 after treatment with CPT or Ac D.


Fig. 3

Tail movement values grouped in intervals for intermediate doses of the different inhibitors.


4 Discussion

We investigated the ability of the comet assay to discriminate between cytotoxins and genotoxins using non-proliferative cells to try to improve it as a biomonitoring test. The drugs selected included the DNA binding chemicals actinomycin D and camptothecin, which are considered genotoxins, and puromycin, cordycepin and fluorodeoxyuridine as cytotoxins.

CPT, a cytotoxic plant alkaloid isolated from Camptotheca acuminata, is an anticancer drug with a broad spectrum of antitumour activity. It is a topoisomerase I poison that stabilizes the DNA–topoisomerase complex (Covey et al., 1989; Hsiang et al., 1985; Hsiang et al., 1989). Ac D is an RNA synthesis inhibitor that binds to DNA and blocks the movement of RNA polymerase I (Jiménez, 1988; Cohen et al., 1998). Puromycin is a protein synthesis inhibitor that causes premature chain termination by acting as an analog of the 3′-terminal end of aminoacyl tRNA (Nathans and Gottlieb, 1967; Garcı́a-Herdugo et al., 1974). Cordycepin, 3′-deoxyadenosine, is an mRNA synthesis inhibitor that blocks elongation of the growing RNA chain (Moreno et al., 1989). Finally, FdU is an antitumour drug that inhibits DNA synthesis by blocking the action of thymidilate synthetase (Escalza et al., 1985; Escalza et al., 1992; Tanaka et al., 1990).

Neither puromycin, cordycepin nor FdU induced DNA migration in the treated quiescent cells, which remained in damage classes 1–3 (Fig. 1B,C,D), like the control cells (Fig. 1A, Fig. 2A). However, the results for Ac D and CPT, which act by binding to DNA, caused DNA migration observed as comet figures; most of the cells treated were distributed among highest damage classes (Fig. 1E,F, Fig. 2B,C). With regard to the average tail moment values (Table 1), only the two DNA binding inhibitors produced results significantly different from controls. Grouping these tail moment values in intervals (Fig. 3) confirmed that only the chemicals classified as genotoxins caused the formation of DNA fragments visible as a comet.

These results support the conclusion of Henderson et al. (1998) that the comet assay distinguishes efficiently between genotoxins and cytotoxins: CPT as well as Ac D provoked DNA strand breaks and comets were observed, while the chemicals that do not bind DNA did not induce the formation of comet figures. The data also agree with results recently published by our group demonstrating that the topo I inhibitor CPT and the RNA synthesis inhibitor Ac D induced DNA strand breaks in unstimulated human white blood cells, whilst Cordy caused no strand breaks in these cells (Daza et al., 2001).

As Tice et al. (2000) reported, this assay can be useful in genetic toxicology: (1) as a potentially high-throughput screening assay, (2) in mechanistic studies to distinguish between genotoxically and cytotoxically induced chromosomal damage, and (3) potentially as a part of a battery of in vitro/in vivo assays. However, the single cell gel assay still needs more studies to confirm its reproducibility and reliability.

The single cell electrophoresis or comet assay has been used widely to measure DNA damage. This assay principally detects single-strand breaks and alkali-labile sites in DNA. It can be also modified to detect DNA crosslinks, but in our opinion it may not be a good monitoring test when non-genotoxins are used.

Normally, such biomonitoring assays are applicable only to cultured cells. Any eukaryotic cell can theoretically be used to test genotoxicity by the comet assay. However, non-proliferating cells may be less prone to the false-positive responses potentially associated with agents that interfere with DNA synthesis. Our contribution to the validation of this test has been to demonstrate its simplicity: we have used cells that do not have to be cultured, and, with only a few drops of blood, we could score and assess the drug effects. It was not necessary to isolate the lymphocytes, so we avoided the effects of Ficoll, which is potentially cytotoxic.

The aim of this work was to confirm that the comet assay is an efficient technique for differentiating the mechanisms of action of different drugs, cytotoxins and genotoxins in this case, using quiescent cells to make the method easier and more rapid. This modification of the protocol, in which neither culture nor lymphocyte isolation was needed, seems superior to the traditional method in that it provides a fast in vitro screening test.

Acknowledgements

We wish to thank Manuel Daza Navarro for checking the translation, Ma Cristina Oettinger for her generous help, and Dr. Santiago Mateos Cordero for his advice. All experiments complied with current Spanish laws.

References

Cohen SM, Cobb, ER, Cordeiro-Stone, M, Kaufman, DG. Identification of chromosomal bands replicating early in the s phase of normal human fibroblasts. Exp Cell Res 1998:245:321-9
Crossref   Medline   

Covey JM, Jaxel, C, Kohn, KW, Pommier, Y. Protein-linked DNA strand breaks induced in mammalian cells by camptothecin, an inhibitor of topoisomerase I. Cancer Res 1989:49:5016-22
Medline   

Daza P, Torreblanca, J, Garcı́a-Herdugo, G, Moreno, FJ. DNA strand breaks induced by the topoisomerase I inhibitor camptothecin in unstimulated human white blood cells. Cell Biol Int 2001:6:8:707-13

Escalza P, Cortés, F, Schwartzman, JB. Induction of sister-chromatid exchanges (SCEs) by 5-fluorodeoxyuridine. The role of 5-bromodoxyuridine incorporated into parental DNA. Mutat Res 1985:151:77-82
Medline   

Escalza P, Daza, P, Piñero, J, Cortés, F. Different effectiveness of 4-nitroquinoline-1-oxide, mitomycin C and ethyl methanesulfonate to induce lesions in DNA leading to sister chromatid exchange throughout successive cell cycles in Chinese hamster ovary cells. Mutagenesis 1992:7:2:137-40
Crossref   Medline   

Fairbairn DW, Olive, PL, O'Neill, KL. The comet assay: a comprehensive review. Mutat Res 1995:339:37-59
Medline   

Festa F, Cristaldi, M, Ieradi, LA, Moreno, S, Cozzi, R. The comet assay for the detection of DNA damage in Mus spretus from Doñana National Park. Environ Res 2003:91:1:54-61
Crossref   Medline   

Garcı́a-Herdugo G, Fernández-Gómez, ME, Hidalgo, J, López-Sáez, JF. Effects of protein synthesis inhibition during plant mitosis. Exp Cell Res 1974:89:336-42
Crossref   Medline   

Hartmann A, Speit, G. Genotoxic effect of chemicals in the single cell gel (SCG) test with human blood cells in relation to the induction of sister-chromatid exchanges (SCE). Mutat Res 1995:346:49-56
Crossref   Medline   

Henderson L, Wolfreys, A, Fedyk, J, Bourner, C, Windebank, S. The ability of the comet assay to discriminate between genotoxins and cytotoxins. Mutagenesis 1998:13:1:89-94
Crossref   Medline   

Hsiang Y, Hertzberg, R, Hecht, S, Liu, LF. Camptothecin induces protein-linked DNA-breaks via mammalian DNA topoisomerase I. J Biol Chem 1985:260:14873-8
Medline   

Hsiang YH, Lihou, MG, Liu, LF. Arrest of replication forks by drug-stabilised topoisomerase I–DNA cleavable complex as a mechanism of cell killing by camptothecin. Cancer Res 1989:49:5077-82
Medline   

Jiménez A. Inhibitors of translation. Trends Biochem Sci 1988:1:28-30

Menke M, Angelis, KJ, Schubert, I. Detection of specific DNA lesions by a combination of comet assay and FISH in plants. Environ Mol Mutagen 2000:35:2:132-8
Crossref   Medline   

Merck O, Speit, G. Detection of crosslinks with the comet assay in relationship to genotoxicity and cytotoxicity. Environ Mol Mutagen 1999:33:167-72
Crossref   Medline   

Moreno FJ, Rodrigo, RM, Garcı́a-Herdugo, G. An experimental approach to nucleolar organization in plant cells: a morphological, cytochemical and quantitative study. J Cell Sci 1989:94:51-9

Moreno FJ, Rodrigo, RM, Garcı́a-Herdugo, G. . Nathans D, Gottlieb D. Antibiotics, vol.1. New York, NY; 1967. p. 259.

Navarrete MH, Carrera, P, De Miguel, M, De La Torre, C. A fast comet assay variant for solid tissue cells. The assessment of DNA damage in higher plants. Mutat Res 1997:389:271-7
Medline   

Olive PL, Wlodek, D, Durand, RE, Banath, JP. Factors influencing DNA migration from individual cells subjected to gel electrophoresis. Exp Cell Res 1992:198:259-67
Crossref   Medline   

Östling O, Johanson, KJ. Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem Biophys Res Commun 1984:123:291-8
Crossref   Medline   

Pastor N, López-Lázaro, M, Tello, JL, Baos, R, Hiraldo, F, Cortés, F. Assessment of genotoxic damage by the comet assay in white storks (Ciconia ciconia) after the Doñana ecological disaster. Mutagenesis 2001:16:219-23
Crossref   Medline   

Pastor N, López-Lázaro, M, Tello, JL, Baos, R, Forero, MG, Hiraldo, F. DNA damage in birds after the Minimo waste spills in southwestern Spain: a comet assay evaluation. J Environ Pathol Toxicol Oncol 2001:20:317-24
Medline   

Singh NP, Mccoy, MT, Tice, RR, Schneider, EL. A simple technique for quantification of low levels of DNA damage in individual cells. Exp Cell Res 1988:175:184-91
Crossref   Medline   

Tanaka Y, Eda, H, Fujimoto, K, Tanaka, T, Ishikawa, T, Ishitsuka, H. Anticachectic activity of 5′-deoxy-5-fluorouridine in a murine tumor cachexia model, colon 26 adenocarcinoma. Cancer Res 1990:50:4528-32
Medline   

Tice RR, Agurelle, E, Anderson, D, Burlinson, B, Hartmann, A, Kobayashi, H. Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 2000:35:206-21
Crossref   Medline   


Received 12 January 2004/17 March 2004; accepted 26 April 2004

doi:10.1016/j.cellbi.2004.04.003


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)