DNA fragmentation induced by all-trans retinoic acid and its steroidal analogue EA-4 in C 2 C 12 mouse and HL-60 human leukemic cells in vitro Raghda S. Alakhras a , Georgia Stephanou a , Nikos A. Demopoulos a *, Konstantinos Grintzalis a , Christos D. Georgiou a and Sotirios S. Nikolaropoulos b ABSTRACT: We have recently shown that retinoic acid induces micronucleation mainly via chromosome breakage (Alakhras et al. Cancer Lett 2011; 306: 15–26). To further study retinoic acid clastogenicity and evaluate DNA damaging potential we investigated the ability of (a) all-trans retinoic acid and its steroidal analogue EA-4 to induce DNA fragmentation by using Comet assay under alkaline unwinding and neutral condition electrophoresis, and (b) the retinoids under study to induce small (0–1 kb) DNA fragments. Two cell lines, C2C12 mouse cells and HL-60 human leukemic cells were used in this study. We found that all-trans retinoic acid and its steroidal analogue EA-4 (a) provoke DNA migration due to DNA fragmentation as it is shown by the increased values of Comet parameters, and (b) induce significantly small-size fragmented genomic DNA as indicated by the quantification of necrotic/apoptotic small DNA segments in both cell systems. A different response between the two cell lines was observed in relation to retinoid ability to increase the percentage of DNA in the tail as well as break DNA in to small fragments. Our findings confirm the ability of retinoic acid to provoke micronucleation by disrupting DNA into fragments, among which small pieces of double-stranded DNA up to 1 kb are identified. Copyright © 2013 John Wiley & Sons, Ltd. Keywords: Retinoids; Comet assay; DNA fragmentation; Small size fragmented genomic DNA; Retinoic acid genotoxicity Introduction The retinoids are natural, synthetic analogues structurally related to vitamin A (Bollag and Holdener, 1992). Among retinoids, all- trans retinoic acid (ATRA) is used to promote differentiation of acute promyelocytic leukemia blasts in patients with the disease. However, its pharmacokinetic properties contribute to a brief duration of remission associated with a progressive decrease in peak plasma concentrations following chronic dosing. This phenomenon prompted many laboratories to synthesize new retinoids, which may provide effective therapy for leukemia as well as for more common malignancies (Delva et al., 1993; Degos et al., 1995; Marcus and Coulston, 2001). EA-4 is a new synthesized steroidal analogue of ATRA with promising antineoplastic activity. It was found that EA-4 inhibited human leukemic cell growth by decreasing the proliferation of human promyelocytic HL-60 cells. This decrease was significant at concentrations higher than 10 –5 M at 24 and 48 h treatments. In addition, EA-4 had no effect on angiogenesis in contrast of ATRA, which induced it as shown by the in vivo chicken embryo CAM assay (Arsenou et al., 2005). Recently, it was observed that ATRA and its analogue EA-4 are cytotoxic and genotoxic to human lymphocytes and C 2 C 12 mouse cells because they reduce cellular proliferation rate and induce micronucleation in both cell systems (Alakhras et al., 2011). Micronucleation is mainly generated via chromosome breakage and to a lesser extent via chromosome delay. Analysis of the mitotic spindle in mouse cells has shown that they affect chromosome orientation during metaphase and induce centrosome defects as well as microtubule arrays disruption in interphase cells. Furthermore, ATRA and EA-4 affect mitotic progression by accumulation of cells in anaphase and telophase with various abnormal figures such as micronucleus inclusion, multiple signals of γ-tubulin, nucleoplasmic bridges and multinucleation (Alakhras et al., 2011). The majority of anticancer drugs have DNA-damaging proper- ties; therefore, patients and employees in drug manufacturing exposed to these drugs are at risk for DNA damage (Maluf and Erdtmann, 2000; Aydemir et al., 2005). Various techniques have been used to evaluate DNA damage. Among these techniques, the single cell gel electrophoresis (SCGE) or Comet assay is a versatile methodology. It has the ability to assess qualitatively *Correspondence to: N. A. Demopoulos, Division of Genetics, Cell and Develop- mental Biology, Department of Biology, University of Patras, 265 00 Patras, Greece. Email: ndemop@biology.upatras.gr a Division of Genetics, Cell and Developmental Biology, Department of Biology, University of Patras, 26 500, Patras, Greece b Laboratory of Medicinal Chemistry, Department of Pharmacy, University of Patras, 26 500, Patras, Greece J. Appl. Toxicol. 2014; 34: 885–892 Copyright © 2013 John Wiley & Sons, Ltd. Research Article Received: 8 April 2013, Revised: 4 June 2013, Accepted: 11 June 2013 Published online in Wiley Online Library: 2 August 2013 (wileyonlinelibrary.com) DOI 10.1002/jat.2908 885