Reproductive Toxicology 29 (2010) 415–420 Contents lists available at ScienceDirect Reproductive Toxicology journal homepage: www.elsevier.com/locate/reprotox Anticancer drug 5-fluorouracil induces reproductive and developmental defects in Caenorhabditis elegans Sandeep Kumar a,c , Caroline Aninat a,c,∗∗ , Grégoire Michaux b,c,d , Fabrice Morel a,c,∗ a INSERM, UMR991, Liver, Metabolisms and Cancer, F-35033 Rennes, France b CNRS UMR6061, Université de Rennes 1, F-35043 Rennes, France c IFR140, Université de Rennes 1, F-35043 Rennes, France d Équipe Avenir INSERM, Université de Rennes 1, F-35043 Rennes, France article info Article history: Received 10 December 2009 Received in revised form 8 February 2010 Accepted 25 February 2010 Available online 4 March 2010 Keywords: Caenorhabditis elegans 5-Fluorouracil Development Egg hatching Vulva abstract In order to examine the chronic effects of anticancer drug 5-fluorouracil (5-FU) on reproduction and development, we exploited Caenorhabditis elegans as a model system. We demonstrate that 5-FU induces cell-cycle arrest and apoptosis of germline cells and reduces by ∼30–40% the number of mitotic nuclei per gonad arm when compared to untreated worms. This drug also affects vulva development, some animals being vulvaless, as well as dysfunction of vulval and egg laying muscles leading to an 8–10 days delay in reproductive time. Interestingly, 5-FU represses levels of mRNA encoding LIN-29, a transcription factor that affects vulva development and egg laying system. Finally, we demonstrate that RNAi-dependent repression of ung-1 gene, which encodes a uracil-DNA glycosylase, partially abolishes 5-FU effects on embryo hatching. Thus, we proposed that C. elegans could be a useful model system for studying the mechanisms by which 5-FU might affect either embryo, adult or organ development. © 2010 Elsevier Inc. All rights reserved. 1. Introduction 5-Fluorouracil (5-FU), an uracil analogue containing a fluorine atom in position C-5, is an antimetabolic drug currently used in the treatment of colorectal, stomach and breast cancer [1]. Several studies have demonstrated 5-FU side-effects including myelotox- icity, gastrointestinal disturbances, cardiotoxicity, hepatotoxicity and neurotoxicity. Although 5-FU has not been investigated extensively in humans to permit an evaluation of its effects on reproduction, animal studies have shown that it also induces chromosomal aberrations, infertility, and teratogenicity [2–5]. In cells, 5-FU is rapidly converted in several active metabolites: fluorodeoxyuridine monophosphate (FdUMP), fluorodeoxyuridine triphosphate (FdUTP) and fluorouridine monophosphate (FUMP). Abbreviations: 5-FU, 5-fluorouracil; AO, acridine orange; BER, base excision repair; DAPI, 4 ′ 6-diamidino-2-phenylindole; DDR, DNA damage response; DPD, dihydropyrimidine dehydrogenase; FdUMP, fluorodeoxyuridine monophosphate; FdUTP, fluorodeoxyuridine triphosphate; FUMP, fluorouridine monophosphate; NGM, nematode growth medium; TS, thymidylate synthase; UNG, uracil-DNA gly- cosylase. ∗ Corresponding author at: INSERM UMR 991, Hôpital Pontchaillou, 35033 Rennes, France. Tel.: +33 2 99 54 74 01; fax: +33 2 99 54 01 37. ∗∗ Corresponding author at: INSERM UMR 991, Hôpital Pontchaillou, 35033 Rennes, France. Tel.: +33 2 99 54 37 37; fax: +33 2 99 54 01 37. E-mail addresses: caroline.aninat@univ-rennes1.fr (C. Aninat), fabrice.morel@inserm.fr (F. Morel). Enzymes involved in the targeted pyrimidine biosynthesis pathway include dihydropyrimidine dehydrogenase (DPD), which converts 5-FU to an inactive metabolite, and thymidylate synthase (TS), which is a major target of 5-FU [6]. Interestingly, Kim et al. [7] have identified single homologs of human DPD and TS in the nematode Caenorhabditis elegans (C. elegans) and showed that the pathway involved in 5-FU metabolism appears to be highly con- served between C. elegans and human at the molecular level [8]. C. elegans is a small, free-living soil nematode (roundworm) and is a genetically well-characterized model organism [9] widely used in developmental biology and genetic studies which presents several advantages: (i) rapid reproduction as self fertile hermaphrodite, (ii) large number of offspring (250–300 progeny), (iii) transparency of body which allows observation of cells in mature and developing animals. Under optimal conditions, the life cycle of C. elegans is about 3 days and is comprised of the embry- onic stage (about 15 h), four larval stages (L1–L4, 2.5 days) and adulthood while the lifespan is approximately 2–3 weeks. Further- more, the embryonic development has been fully described from the moment an egg is fertilized until it hatches [10]. Recently, Kim et al. [7] have demonstrated that 5-FU induces germ cell death in C. elegans [11]. Furthermore, Dengg et al. [12] have demonstrated that depletion of dUTPase in C. elegans leads to an increase of uracil incorporation into DNA which interfered with germline and vulva development. Therefore, our study aimed at investigating the effects of 5-FU on reproduction and development of C. elegans by analyzing vulva development, body length and egg 0890-6238/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.reprotox.2010.02.006