Elsevier

Toxicology

Volume 207, Issue 2, 14 February 2005, Pages 331-338
Toxicology

The effects in mice of combined treatments to X-rays and antineoplastic drugs in the Comet assay

https://doi.org/10.1016/j.tox.2004.10.002Get rights and content

Abstract

The Comet assay is a rapid, easy and reproducible method to detect genotoxic activity of chemical and physical agents in vitro and in vivo. In the present study the effects of exposure to irradiation or chemicals: cyclophosphamide (CP) and mitomycin C (MMC) or combined exposure to low doses of both agents (0.25 Gy + 3.15 mg/kg bw CP and 0.25 Gy + 0.25 mg/kg bw MMC) were examined for the induction of DNA damage in the Comet assay measured simultaneously in somatic (bone marrow lymphocytes) and haploid germ cells. The male mice were treated in vivo and sacrificed at 24 h after exposure.

The percentage contents of DNA in the “comet tail” increased with increasing doses of X-rays and chemicals. After combined exposure to X-rays and CP and to X-rays and MMC weak increases of DNA damage in bone marrow lymphocytes and in germ cells were observed by comparison with the results obtained for each agent acting alone.

There were slightly different responses in bone marrow lymphocytes and in germ cells, but effects were observed over a similar dose range.

Introduction

The single-cell gel electrophoresis (SCGE), Comet assay is a rapid, easy and reproducible method to detect genotoxic activity of chemical and physical agents in vitro and in vivo. This technique is sensitive enough to detect low levels of DNA damage and repair of damage in individual cells and cells are examined using a fluorescence microscope. Cells look like comets with a bright fluorescent head and tail, the length and intensity of which are related to the number of DNA strand breaks. The Comet assay can potentially measure DNA lesions in any organ or tissue even in the absence of mitotic activity. Although, a perfect correlation between chemicals positive in this test and carcinogenicity is not expected, the Comet assay can be used to identify possible human mutagens and carcinogens (Anderson et al., 1998, Tice et al., 2000). The SCGE assay is used in genetic toxicology, radiation biology, oncology and in environmental research.

Radiation comes from natural and man-made sources. Radiation is a well-known mutagenic and carcinogenic agent. Exposure to X-rays is possible in cancer therapy and during diagnostic investigations. In the nuclear power industry employees can accidentally receive relatively large doses of radiation, but medical personnel are generally exposed to relatively low doses. Occupational exposure to ionizing radiation may result in DNA damage leading to chromosome aberrations (Kubelka et al., 1992a, Pohl-Ruling, 1992, Garaj-Vrhovac et al., 1997).

Cyclophosphamide (CP) and mitomycin C (MMC) are anticancer agents, capable of inducing various types of primary DNA damage, gene mutations and chromosomal aberrations. They can be used to improve the effect of radiotherapy. The doses used for therapy are usually high and repeated several times. The doses for radiotherapy usually vary from 1.5 to 13.8 Gy daily, with the total dose ranging from 30 to 64 Gy (Wada et al., 2004, Budach et al., 2002). The doses of CP vary from 200 to 1000 mg/m2 over several days (Vinolas et al., 2002, Manova et al., 2000). The doses of MMC vary from 2 to 10 mg/m2 (Yamao et al., 2001, Hartmann et al., 2003) applied usually at different levels on respective days.

Except for patients who are treated with both chemical and physical agents, medical employees such as nurses and pharmacy personnel might be exposed to low doses of X-rays and antineoplastic drugs. They may show symptoms of anticancer drug overexposure, especially manifested in the changes of blood, like anemia, thrombocytopenia, neurogenia and immunodeficiency (Bruick, 1979). Some papers have described the biological effects of antineoplastic drugs in nurses. After exposure to anticancer drugs, chromosomal aberrations, sister chromatid exchanges, micronuclei or DNA damage in peripheral blood lymphocytes have been observed (Oestreicher et al., 1990, Thringer et al., 1991, Machado-Santelli et al., 1994, Fuchs et al., 1995, Fucic et al., 1998, Undeger et al., 1999).

In the present assay effects of exposure to irradiation or anticancer drugs (CP and MMC) on the simultaneous induction of DNA damage in somatic (bone marrow lymphocytes) and haploid germ cells were examined on the Comet assay, as well as the effects of combined exposures to low doses of X-rays and each of the drugs in both types of cells. Investigations of the effects of combined X-rays–CP and X-rays–MMC exposure in the Comet assay have not been reported before.

Section snippets

Materials and methods

Pzh:SFIS male mice were obtained from the Laboratory of Animal Breeding of the National Institute of Hygiene. Animals were housed in plastic cages in a room designed for the control temperature and humidity, and light cycle (12 h light and 12 h dark). The standard rodent diet and tap water were available ad libidum. Eight to 12 week old animals were assigned randomly to either control or each of the exposed groups. All animals received a minimum of 5 days of acclimatisation before the start of

Results

All assays have been repeated five times. Fig. 1 presents the mean results of five animals per dose. The results of the statistical analysis are shown in Table 1.

In irradiated animal groups, the percentage contents of DNA in the “comet tail” increased non-linearly with dose, in somatic as well as in germ cells. The results of 0.5, 1.00 and 2.00 Gy were statistically significantly different from controls in both types of cells, but in lymphocytes greater differences in DNA migration between

Discussion

There have not been many studies performed to investigate the effects of DNA damage measured in the Comet assay after in vivo exposure of animals. Carrera et al. (1998) found that the level of DNA damage in mouse liver produced by γ-irradiation was highly statistically significant at doses of 0.5 and 1.00 Gy. Mendiola-Cruz and Morales-Ramirez (1999) observed the induction of comets in murine peripheral blood lymphocytes of mice exposed to 1.00 Gy of γ-rays in a 127Cs source. Haines et al. (2001)

Acknowledgments

The author acknowledges the help of Prof. Diana Anderson (University of Bradford) for reading this manuscript and useful discussion. The author also appreciates the technical assistance of Anna Sawicka and Teresa Olejniczak.

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