Mutation Research 738–739 (2012) 1–11 Contents lists available at SciVerse ScienceDirect Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis jo ur n al hom ep a ge: www.elsevier.com/locate/molmut C om mun i ty a ddress: www.elsevier.com/locate/mutres Mechanisms involved in alternariol-induced cell cycle arrest A. Solhaug a,∗ , L.L. Vines b , L. Ivanova a , B. Spilsberg a , J.A. Holme c , J. Pestka b , A. Collins d , G.S. Eriksen a a Norwegian Veterinary Institute, Oslo, Norway b Michigan State University, Department of Food Science and Human Nutrition, East Lansing, MI, USA c Norwegian Institute of Public Health, Division of Environmental Medicine, Oslo, Norway d University of Oslo, Department of Nutrition, Faculty of Medicine, Oslo, Norway a r t i c l e i n f o Article history: Received 3 July 2012 Received in revised form 31 August 2012 Accepted 21 September 2012 Available online 29 September 2012 Keywords: Mycotoxins Alternariol DNA damage ROS p53 Immunotoxicity a b s t r a c t Alternariol (AOH), a mycotoxin produced by Alternaria sp, is often found as a contaminant in fruit and cereal products. Here we employed the murine macrophage cell line RAW 264.7 to test the hypothesis that AOH causes toxicity as a response to DNA damage. AOH at concentrations of 15–30 M almost completely blocked cell proliferation. Within 30 min treatment, AOH (30 M) significantly increased the level of reactive oxygen species (ROS). Furthermore, DNA base oxidations as well as DNA strand breaks and/or alkaline labile sites were detected by the comet assay after 2 h exposure of AOH. Cell death (mostly necrosis) was observed after prolonged exposure to the highest concentration of AOH (60 M for 24 and 48 h) in our study. The DNA damage response involved phosphorylation (activation) of histone H2AX and check point kinase-1- and 2 (Chk-1/2). Moreover, AOH activated p53 and increased the expression of p21, Cyclin B, MDM2, and Sestrin 2; likewise the level of several miRNA was affected. AOH-induced Sestrin 2 expression was regulated by p53 and could at least partly be inhibited by antioxidants, suggesting a role of ROS in the response. Interestingly, the addition of antioxidants did not inhibit cell cycle arrest. Although the formation of ROS by itself was not directly linked cell proliferation, AOH-induced DNA damage and resulting transcriptional changes in p21, MDM2, and Cyclin B likely contribute to the reduced cell proliferation; while Sestrin 2 would contribute to the oxidant defense. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Mycotoxins, biologically active secondary fungal metabolites found as contaminants of food and feed, pose a major risk for dis- ease in humans and animals [1]. The fungus Alternaria sp. occurs frequently in grains and a negative interaction between Fusarium Abbreviations: H2DFDA, 2  ,7  -dichlorodihydrofluorescein diacetate; JC-1, 5  ,6,6  -tetrachloro-1,1  ,3,3  -tetraethylbenzimidazolylcarbocyanineiodide; 7-ADD, 7-amino actinomycin; 8-oxoGua, 8-oxo-7,8-dihydroguanine; AOH, alternar- iol; AME, alternariol monomethyl ether; ANOVA, analysis of variance; AP, apurinic/apyriminic; Asc, ascorbic acid; ATM, ataxia telangiectasia mutated; ATR, ATM-Rad3-related; BSA, bovine serum albumin; BRCA, breast cancer; Chk-1/2, checkpoint kinase 1/2; DHE, dihydroethidium; DSBs, DNA double stranded breaks; SSBs, DNA single stranded breaks; fpg, formamidopyrimidine DNA glycosylase; HRP, horse radish peroxidase; LMPA, low melting point agarose; miRNA, microRNA; MDM2, murine double minute 2; NAC, N-acetyl-cysteine; NMPA, normal melt- ing point agarose; nn, not noteworthy; PFA, paraformaldehyde; PBS, phosphate buffered saline; pft-, pifithrin-; PCNA, proliferating cell nuclear antigen; PI, propidium iodide; qRT-PCR, quantitative real time polymerase chain reaction; ROS, reactive oxidant species; TBST, TBS-tween; TeA, tenuazonic acid; TBS, TRIS-buffered saline. ∗ Corresponding author at: Norwegian Veterinary Institute, P.O. Box 750 Sentrum, 0106 Oslo, Norway. Tel.: +47 23216234; fax: +47 23216201. E-mail address: Anita.Solhaug@vetinst.no (A. Solhaug). graminearum and Alternaria spp have been indicated [2]. Alternaria species, including A. alternata, A. citrii and A. tenuissima, produce mycotoxins such as alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TeA) that have been reported in grain as well as fruits [1]. It has been shown that extracts of A. alter- nata are genotoxic and mutagenic in vitro [3–5]. However, no cancer studies of Alternaria toxins in experimental animals have been car- ried out [6]. Although the mutagenicity of A. alternata extracts is well established, less is known about the toxicity of the pure tox- ins produced by this mold. In vitro studies have reported that AOH (Fig. 1) causes DNA damage and is more cytotoxic than both AME and TeA [7,8]. AOH have been reported to be mutagenic in both V79 and mouse lymphoma L5178Y assays at concentrations 10 M and higher [3]. In addition, AOH induces DNA strand breaks in cell-free [9] and cell culture conditions [7,10]. Interestingly, AOH also has the potential to act as a topoisomerase II poison [7], which might contribute to its ability to cause DNA breaks. Some studies have reported effects on cell proliferation and cell cycle arrest. In pri- mary porcine endometrial cells, AOH induces a cell cycle arrest in the G 0 /G 1 phase [11], while Chinese hamster V79 lung fibroblasts, mouse lymphoma cells [3] as well as Hepa1c1c7 and Hepac1c12 cells [12] have been shown to be arrested in the G 2 phase. The effects of AOH in the in vitro experiments described above are all observed in the micromolar range, typical between 5 and 60 M. 0027-5107/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mrfmmm.2012.09.001