Parallel evaluation of doxorubicin‐induced genetic damage in human lymphocytes and sperm using the comet assay and spectral karyotyping

Mutagenesis vol. 19 no. 4 pp. 313±318, 2004 DOI: 10.1093/mutage/geh032 Parallel evaluation of doxorubicin-induced genetic damage in human lymphocytes and sperm using the comet assay and spectral karyotyping1 A.Baumgartner1,2, T.E.Schmid1,3, E.Cemeli1 and D.Anderson1,4 1Department of Biomedical Sciences, University of Bradford, Bradford BD7 1DP, UK, 2GSF Research Center, Institute of Molecular Radiobiology, 85764 Neuherberg, Germany and 3School of Public Health, University of California in Berkeley, Berkeley, CA 94720, USA In recent years, two techniques for detecting genetic damage in the whole genome have gained importance: the alkaline comet assay, to detect DNA damage such as strand breaks and alkali-labile sites, and a multicolour FISH method, spectral karyotyping (SKY), to identify chromosomal aberrations simultaneously in all metaphase chromosomes. In the present study, the induction of DNA damage in human sperm and lymphocytes in vitro has been studied employing an anticancer drug, doxorubicin (DX). An increase in DNA damage was observed with the comet assay as the median per cent head DNA of sperm signi®cantly decreased from 82.07 and 85.14% in the untreated control groups to 63.48 and 72.52% at doses of 0.8 mM DX. At 1.6 mM the percentage declined to 60.96% (the corresponding tail moment increased from 4.42 to 12.19). In stimulated lymphocytes, a signi®cant increase was observed in tail moment, from 0.72 and 0.53 in controls to 15.17 and 12.10 at 0.2 mM DX, continuing at the same level to a ®nal concentration of 1.6 mM. Structural aberrations found in the parallel SKY study in stimulated lymphocytes at 0.2 mM DX consisted of 14% chromatid-type and 2% chromosome-type aberrations; none were found in controls. The SKY results correlate very well with the ®ndings of the comet assay in lymphocytes where DNA damage was observed at similar doses. This study is the ®rst reporting use of the comet assay and SKY analysis in parallel after chemical treatment. The potential of the two techniques together is evident, as they represent a set of assays feasible for evaluating damage in human somatic and germ cells after chemical treatment (i) by direct observation of two different endpoints, detecting general DNA damage and chromosomal aberrations and (ii) by extrapolation from lymphocytes to sperm, which provides a `parallelogram' approach in human cells. Introduction The importance of assessing genotoxicity in human somatic and germ cells will continue to have an important role in the future, particularly with concerns about the detrimental effects on reproduction in terms of male infertility and sperm count (Medical Research Council, 1995). The single cell gel electrophoresis (comet) assay has already been extensively used to detect DNA damage in the whole genome in single cells as double- and single-strand breaks and alkali-labile sites (Singh et al., 1988; Anderson et al., 1998). However, a combination of the comet assay with a multicolour ¯uorescence in situ hybridization (FISH) method, spectral karyotyping (SKY), which detects chromosomal aberrations in all human metaphase chromosomes simultaneously (SchroÈck et al., 1996), allows a different type, yet detailed, assessment of induced damage in the whole genome. In the comet assay, induced DNA damage is evaluated after single cell gel electrophoresis by measuring the tail moment as the product of per cent tail DNA multiplied by the tail length of the comet and the per cent head DNA. After alkaline lysis, damaged DNA originating from DNA strand breaks and alkalilabile sites thereby pass out of the nuclei moving towards the anode along the electrical ®eld and form comet-like structures (Tice et al., 2000; Olive, 2002). In contrast, with SKY, speci®c chromosomal damage can be evaluated, for numerical and structural aberrations in all 46 human chromosomes simultaneously by employing FISH with DNA probes labelled with a combination of ®ve different ¯uorochromes. Thus, each chromosome has a unique ¯uorescence spectrum, enabling computer software to classify all 24 different human chromosomes (22 pairs plus X and Y) ef®ciently. The threshold for detecting translocation fragments is ~10±20 Mb (Gray and Collins, 2000; Wang et al., 2002). In contrast to classical methods of chromosome aberration detection, where Giemsa banding (see for example Ronne, 1990) has mainly been used, SKY is able to detect hidden aberrations and uncharacterized marker chromosomes throughout the whole genome by evaluating all chromosomes from their distinct colour (Mohr et al., 2000). When cancer has been diagnosed in a patient, various treatments using multiple combinations of chemotherapeutic agents are possible, depending on the type of cancer. Some of these anticancer agents speci®cally act on dividing cells during mitosis by inhibiting cell division (reviewed in Stewart et al., 2003), while others act non-speci®cally (reviewed in Blagosklonny, 2003). As a consequence, they cause apoptosis and/or necrosis, subsequently leading to cell death. Proliferating cells are more likely to die as a result of the damage induced, but cells surviving the exposure may carry mutations and chromosomal aberrations. To evaluate the combination of comet and SKY in human somatic and germ cells, doxorubicin (DX), a well-known anticancer drug, was employed. DX, formerly known as adriamycin, is an anthracycline antibiotic produced by the fungus Streptomyces peucetius. DX treatment at therapeutic doses leads to the induction of CD95-ligand (CD95-L). CD95-L can mediate cell death (apoptosis) by cross-linking the CD95 receptor (CD95) (Friesen et al., 1999). DX is also able to inhibit reverse transcriptase (Tomita and Kuwata, 1976) and RNA synthesis (Li and Yu, 1993). However, at the DNA level, three major mechanisms of action have been identi®ed for DX (Cummings 4To whom correspondence should be addressed. Tel: +44 1274 23 3569; Fax: +44 1274 30 9742; Email: Mutagenesis vol. 19 no. 4 ã UK Environmental Mutagen Society 2004; all rights reserved. 313 A.Baumgartner et al. et al., 1991): binding DNA effectively by intercalation of the anthracycline portion; causing DNA damage via the production of free radicals from reactive oxygen species (ROS); stabilizing the topoisomerase II cleavage complex, which is critical for DNA function. As a consequence of these multiple effects, single- and double-strand breaks are introduced into the DNA (Cummings et al., 1991). It was shown that DX induces chromosome aberrations in murine bone marrow cells and in spermatocytes and that chromosome aberrations were sustained in the surviving spermatogonial cells (Au and Hsu, 1980). DX also increased the frequency of meiotic micronuclei in male rats (Laehdetie et al., 1983), which showed impaired fertility after being treated. Decreased weights of the genital organs, an extremely decreased number of sperm, low sperm motility, a low implantation rate and a decreased number of live foet (...truncated)