miRNAs as tools for sensing early organ toxicity

Abstracts / Toxicology Letters 205S (2011) S19–S35

Reference Worek, F., 2010. Chem. Biol. Interact. 187, 259–264. Wagner, S., 2010. PLoS One 5, e14213.

doi:10.1016/j.toxlet.2011.05.122

W10 miRNAs in Drug Safety Assessment and Their Implication for Drug Development

W10-1 MicroRNAs in the cytotoxic response M. Bushell MRC Toxicology Unit, Leicester, UK Over the past 8 years several lines of compelling evidence have indicated that microRNAs are critical downstream effectors of classic oncogene/tumour suppressor networks. The archetypal examples of oncogene and tumour suppressor microRNAs are the miR-17-92 (oncomir 1) polycistron and the miR-34 family, respectively. Whilst the involvement of these two opposing families of microRNAs in oncogenesis has been known for some time, the mRNA targets through which they exert their phenotypes are only just beginning to be uncovered. Moreover, several recent reports have demonstrated that the relevant physiological targets of certain individual microRNAs are actually fairly limited, with repression of just one or two major targets being sufficient to explain the observed phenotype. We will present our recent data examining the role of microRNAs in the response to cytotoxic insult and how they exert their effects, in particular, focusing on how these mechanisms are deregulated in human cancers. doi:10.1016/j.toxlet.2011.05.124

W10-2 miRNAs as tools for sensing early organ toxicity C. Goldring MRC Centre for Drug Safety Science, Department of Pharmacology & Therapeutics, Liverpool, UK New biomarkers of tissue injury are urgently required in the clinic and in preclinical pharmaceutical evaluation. Novel liver injury biomarkers may be particularly important. MicroRNAs (miRNAs) potentially represent a new class of biomarkers which are stable and sensitive, and some of these molecules display a high degree of tissue-selective expression. In this presentation, the background to the use of miRNAs to detect organ toxicity will be described and data will be presented from studies in our group on miRNA markers of liver and kidney injury. We see a significant early rise in miR-122 in the plasma of mice treated with a toxic dose of acetaminophen. This precedes a rise in serum ALTs. Concentrations of miR-122 are significantly higher (252-fold increase, P < 0.0001) in the serum of patients with acetaminophen-induced liver injury (n = 53) compared to a control cohort (n = 25). Serum miR-192 is also significantly higher in the patient cohort (65-fold increase, P < 0.0001). Non-hepatic serum miRNA, miR-1 (enriched in heart) shows no difference between the overdose and control cohorts. Our data show that serum miR-122 significantly correlates with peak ALT levels (r2 = 0.47; P < 0.01) but does not correlate with

S31

prothrombin time or serum creatinine. Serum miR-122 levels are 110% higher in patients who had poor outcome but this is not statistically significant (P = 0.39). In urinary exosomes, miR-192 and miR-122 are detectable but this does not correlate with kidney function. This work provides evidence of miRNAs as biomarkers of DILI in humans, and provides a platform for further pre-clinical validation studies and for clinical qualification. doi:10.1016/j.toxlet.2011.05.125

W10-3 Integrated miRNA microarray analysis in the context of non-genotoxic rodent hepatocarcinogenesis S. Plummer 1,∗ , D. Dan 1 , J. Quinney 1 , C. Vacchi-Suzzi 2 , P. Couttet 2 CXR Biosciences Ltd, Dundee, UK, 2 Investigative Toxicology, Novartis Institutes for Biomedical Research, Basel, Switzerland 1

Development of biomarkers for risk assessment of xenobiotic potential to induce non-genotoxic liver carcinogenesis (NGC) in rodents is an important challenge for the drug/chemical industry. To address this we performed integrated expression and microRNA (miRNA) array analysis in the context of mice doubly humanised (h) and nulled (KO) for the constitutive androstane (CAR) and the pregnane X (PXR) receptors. Wild type (WT), CARKOPXRKO and hCARhPXR mice were treated with phenobarbital (IP) at 80 mg/kg, for 4 days. Liver RNA was subjected to expression (mRNA) and miRNA microarray analysis using Agilent mouse whole genome and Agilent Mouse miRNA microarrays. Differentially expressed genes (DEG) and significantly altered miRNA (miRNA signatures), relative to genotype controls, were identified using GeneSpring and Rosetta Resolver softwares, respectively. Integrated analysis of DEG and miRNA signatures was performed using GeneSpring (GS) and Ingenuity Pathways Analysis (IPA) in tandem. Up-regulated and down-regulated miRNA signatures were linked in GS with expression array targets via targetscan database analysis. To maximise utility of putative NGC biomarkers we focussed on pathways relevant to NGC. Previous work has shown that only WT mice undergo cell proliferation in response to this treatment. Down-regulated miRNAs in the WT data set were associated with ribonucleotide reductase M2 (Rrm2) which was up-regulated (3.6 fold). This change occurred in WT but not in CARKOPXRKO and hCARhPXR mice and alterations to miRNAs linked with Rrm2 concurred across the genotypes. Hence integrated microarray analysis is useful in the definition of new mechanistic insights for NGC which may prove useful as biomarkers. doi:10.1016/j.toxlet.2011.05.126

W10-4 Integrating miRNAs within current safety assessment: Future perspectives A. Boobis Medicine, Imperial College London, London, UK MicroRNAs (miRNAs) are negative regulators of gene expression at the post-transcriptional level. They have multiple targets and are involved in a range of biological processes, including development, cell proliferation, differentiation and apoptosis. This has opened up exciting prospects in both diagnosis and treatment of human diseases, including cancer. Proof-of-concept studies have