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.
Tail movement values grouped in intervals for intermediate doses of the different inhibitors.
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.
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.
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Received 12 January 2004/17 March 2004; accepted 26 April 2004