Mutation Research 717 (2011) 116–128 Contents lists available at ScienceDirect Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis journa l h o me pa g e: www.elsevier.com/locate/molmut Co mm unit y add re ss: www.elsevier.com/locate/mutres Mutagens interfere with microRNA maturation by inhibiting DICER. An in silico biology analysis Matteo Ligorio a,b , Alberto Izzotti a,∗ , Alessandra Pulliero a , Patrizio Arrigo c a Department of Health Sciences, University of Genoa, Via A. Pastore 1, 16132 Genoa, Italy b Department of Surgery, Massachusetts General Hospital, Harvard University Medical School, Boston, MA, USA c National Research Council CNR Institute for Macromolecule Research ISMAC, Genoa, Italy a r t i c l e i n f o Article history: Received 20 January 2011 Received in revised form 25 July 2011 Accepted 30 July 2011 Available online 26 August 2011 Keywords: DICER microRNA Bioinformatics Mutagens Epigenetic damage a b s t r a c t Exposure to environmental mutagens results in alteration of microRNA expression mainly oriented towards down-regulation, as typically observed in cigarette smoke. However, the molecular mechanism triggering this event is still unknown. To shed light on this issue, we developed an ‘in silico’ analysis testing 25 established environmental mutagens (polycyclic aromatic hydrocarbons, heterocyclic com- pounds, nitrosoamines, morpholine, ethylnitrosurea, benzene derivatives, hydroxyl amines, alkenes) for their potential to interfere with the function of DICER, the enzyme involved in the cytoplasmic phase of microRNA maturation. In order to analyse the binding affinity between DICER and each mutagen, the three-dimensional bioinformatic structures of DICER-RNase III domains and of mutagens have been constructed. The binding affinity of mutagens for each DICER’s RNase III domain was estimated by cal- culating the global contact-energy and the number of intermolecular contacts. These two parameters reflect the stability of the DICER–mutagen complexes. All the 25 mutagens tested form stable complexes with DICER, 20 of which form a complex with DICER A domain, that is more stable than those formed by DICER with its natural substrate, i.e. double strand short RNAs. These mutagens are benzo(a)pyrene diol epoxide, nitroimidazoles, fluorenes, naphthalene, morpholine, stilbenes, hydroxylamines, fecapentenes. In the case of exposure to mutagen mixtures (benzo(a)pyrene-diolepoxide and 4-acetylaminostilbene), synergistic or less than addictive effects occur depending on the docking order of the compounds. A group of 8 mutagens with the highest ability to interfere with this DICER function, was identified by hierarchical cluster analysis. This group included 1-ethyl-1-nitrosourea and 4-nitrosomorpholine. Herein, presented data support the view that mutagens interfere with microRNA maturation by binding DICER. This finding sheds light on a new epigenetic mechanism exerted by environmental mutagens in inducing cell damage. © 2011 Elsevier B.V. All rights reserved. 1. Introduction MicroRNAs (miRNAs) are short non-coding RNAs present in human cells which are directly related to the regulation of gene expression [1]. Their role as inhibitors of messenger RNAs (mRNAs) translation [2] and their involvement in epigenetic regulation mechanisms [3] are currently the hypothesized mechanism to explain their influence in critical biological processes, such as development [4], differentiation [5], proliferation [6] and apopto- Abbreviations: miRNA, microRNA; mRNA, messanger RNA; pri-miRNA, primary micro-RNA; pre-miRNA, micro-RNA precursor; endoND, endonuclease domain; dsRBD, dsRNA-binding domain; PAZ, Piwi Argonaute Zwille; cid, PubChem code; NoHA, number of heavy atoms; TPS, topological polar surface; GCE, global contact energy; NoCs, non-covalent intermolecular contacts; BPDE, benzo(a)pyrene-diol- epoxide; ACTMS, 4-acetylaminostilbene. ∗ Corresponding author. Tel.: +39 0103538522; fax: +39 0103538504. E-mail address: izzotti@unige.it (A. Izzotti). sis [7]. As masters of gene expression regulation, microRNAs play a critical role in the pathogenesis of many different human dis- eases, including cancer [8]. Human cancers are characterized by a prevalent microRNA down-regulation and this down-regulation is causally linked with tumorigenesis [9]. The development of can- cer has been linked to dysregulation of the enzymes involved in microRNA biogenesis [10]. MicroRNA alteration has been recog- nized as an early pathogenic step of the carcinogenesis process. Recently, prevalent microRNAs down-regulation in the lung cells of healthy rats [11] and mice [12] when exposed to cigarette smoke has been demonstrated. This down-regulation could be caused by the interaction between chemical agents, present in cigarette smoke, and enzymes involved in microRNA biogenesis. To test this hypothesis, we created an “in silico” model to explore if some well-known mutagens are able to bind DICER interfering with its catalytic activity. The same bioinformatics model was established to explore if in a mixture of mutagens the concurrent binding of two different mutagens, at the same DICER domain, can have 0027-5107/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2011.07.020