The effects in mice of combined treatments to X-rays and antineoplastic drugs in the Comet assay
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.
References (54)
- et al.
The effect of various antioxidants and other modifying agents on oxygen-radical-generated DNA damage in human lymphocytes in the Comet assay
Mutat. Res.
(1994) - et al.
An investigation of bone marrow and testicular cells in vivo using the comet assay
Mutat. Res.
(1996) - et al.
Cytogenetic analysis of peripheral blood lymphocytes of occupational workers exposed to low levels of ionising radiation
Mutat. Res.
(1999) - et al.
Cytogenetic analysis of lymphocytes from hospital workers, occupationally exposed to low level of ionizing radiation
Mutat. Res.
(1993) - et al.
In vivo response of mouse liver to γ-irradiation assessed by the comet assay
Mutat. Res.
(1998) - et al.
DNA damage in nurses handling antineoplastic agents
Mutat. Res.
(1995) - et al.
Cytogenetic consequences after occupational exposure to antineoplastic drugs
Mutat. Res.
(1998) - et al.
Increased levels of comet-detected spermatozoa DNA damage following in vivo isotopic- or X-irradiation of spermatogonia
Mutat. Res.
(2001) - et al.
Enhanced frequency of chromosome aberrations in workers occupationally exposed to diagnostic X-rays
Mutat. Res.
(1991) - et al.
The value of cytogenetic monitoring versus film dosimetry in the hot zone of a nuclear power plant
Mutat. Res.
(1992)
Biomonitoring of nurses handling antineoplastic drugs
Mutat. Res.
Follow-up of the genetic damage in lymphocytes of pharmacists and nurses handling antineoplastic drugs evaluated by cytokinesis-block micronuclei analysis and single cell gel electrophoresis assay
Mutat. Res.
Repair kinetics of gamma-ray induced DNA damage determined by the single gel electrophoresis assay in murine leukocytes in vivo
Mutat. Res.
Chromosome and SCE analysis in peripheral lymphocytes of person occupationally exposed to cytostatic drugs handled with and without use of safety covers
Mutat. Res.
Sister chromatid exchanges in lymphocytes of nurses handling antineoplastic drugs
Toxicol. Lett.
Simple detection of chemical mutagens by the alkaline single cell gel electrophoresis (Comet) assay in multiple mouse organs (liver, lung, spleen, kidney, and bone marrow)
Mutat. Res.
A simple technique for quantitation of low levels of DNA damage in individual cells
Exp. Cell Res.
Assessment of DNA damage in nurses handling antineoplastic drugs by the alkaline COMET assay
Mutat. Res.
Comparison of alkaline single cell gel (Comet) and peripheral blood micronucleus assay in detecting DNA damage caused by direct and indirect acting mutagens
Mutat. Res.
A multi-institutional reteospective analysis of external radiotherapy for mucosal melanoma of head and neck in Northern Japan
Int. J. Radiat. Oncol. Biol. Phys.
Methods and concepts in detecting abnormal reproductive outcomes of paternal origin
Reprod. Toxicol.
Phase I–II study of irinotecan combined with mitomycin C in patients with advanced gastric cancer
Ann. Oncol.
Developmental capacity of damaged spermatozoa
Hum. Reprod.
Relative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa
Biol. Reprod.
Comet assay response as indicators of carcinogenic exposure
Mutagenesis
The influence of radiation on fertility in man
Br. J. Radiol.
The cellular basis of cancer chemotherapy
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