Toxicology Letters 145 (2003) 303–311 Toxic interaction between hydroxyurea and 1--d-arabino-furanosylcytosine on the DNA of a human hepatoma cell line (HEPG2) Isabelle Severin, Martine Padieu, Jean-Claude Lhuguenot, Marie-Christine Chagnon ∗ UMR (1234), Toxicologie Alimentaire, INRA/ENSBANA 1, Esplanade Erasme, 21000 Dijon, France Received 1 April 2003; received in revised form 24 July 2003; accepted 19 August 2003 Abstract Hydroxyurea (HU) and 1--d-arabino-furanosylcytosine (AraC) are two compounds used to inhibit DNA repair in the comet assay and thereby increase its sensitivity. We used RNA synthesis and comet assays to assess the cytotoxic and genotoxic effects of HU and AraC in the HepG2 cells after 1, 5, or 21h of exposure to concentrations used to inhibit DNA repair. HU was genotoxic between 2 and 10 mM after 1 h of exposure and cytotoxic after 21 h. The presence of AraC (10, 50, or 100 M) increased the DNA damage caused by HU (10 mM) suggesting a potentiation of the genotoxic effect. The interaction between the two inhibitors started after 5 h but was not dependent on the concentrations of AraC. Consequently, careful attention is required when employing a combination of HU and AraC, as their mechanisms of action could interfere with the interpretation of the data from genotoxicity assays. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: HepG2 cell line; Hydroxyurea; 1--d-Arabino-furanosylcytosine; Interaction; Comet assay; Uridine uptake assay 1. Introduction Since its development, the single-cell gel elec- trophoresis (SCGE), or comet, assay has rapidly be- come one of the most widely used methods in genetic toxicology. The alkaline version of the assay (Singh Abbreviations: AraC, 1--d-arabino-furanosylcytosine; DMEM, Dulbecco’s modified eagle’s medium; DMSO, dimethyl sulfoxide; dNTP, desoxynucleotide triphosphate; FBS, fetal bovine serum; HDC, highly damaged cells; HU, hydroxyurea; NER, nucleotide excision repair; UCs, undamaged cells ∗ Corresponding author. Tel.: +03-80-39-66-37; fax: +03-80-39-66-41. E-mail address: mcchagn@u-bourgogne.fr (M.-C. Chagnon). et al., 1988; Tice et al., 2000), in some cases coupled to lesion-specific glycosylase assays (Dusinska and Collins, 1996), allows the detection and quantifica- tion in single-cells of a variety of DNA lesions (DNA single-strand breaks (SSB), alkali-labile sites, and SSB associated with incomplete excision repair site). The comet assay has been applied to a variety of fields of study, including DNA repair, mechanisms of mutage- nesis, ecogenotoxicology, tissue specificity (Fairbairn et al., 1995) and the biomonitoring of human exposure to genotoxic agents (Kassie et al., 2000). The comet assay has numerous advantages over other genotoxi- city tests: accuracy, flexibility, low cost, ease of use and rapidity. It can also be successfully applied to 0378-4274/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2003.08.003