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AbstractIn 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.
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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 1 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 (500
2.3 DNA–comet assay (alkaline) Regular slides were coated with a 1% solution of standard agarose in distilled water by immersing vertically for 2 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 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 Table 1. Average tail moment values for the different inhibitors Full-size table (8K)
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. 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ISSN Print: 1065-6995
ISSN Electronic: 1095-8355 Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB) |
