Amiodarone biokinetics, the formation of its major oxidative metabolite and neurotoxicity after acute and repeated exposure of brain cell cultures Giuliana Pomponio a,d,1 , Marie-Gabrielle Zurich b,1 , Luise Schultz c,1 , Dieter G. Weiss c , Luca Romanelli d , Alexandra Gramowski-Voss c , Emma Di Consiglio a,⇑ , Emanuela Testai a a Mechanism of Toxicity Unit, Environment and Primary Prevention Department, Istituto Superiore di Sanità, Rome, Italy b Department of Physiology, University of Lausanne, Lausanne, Switzerland c Department of Animal Physiology, Institute of Biological Sciences, University of Rostock, Rostock, Germany d Department of Physiology and Pharmacology ‘‘V. Erspamer’’, Università Sapienza, Rome, Italy article info Article history: Received 25 July 2014 Accepted 26 January 2015 Available online xxxx Keywords: Amiodarone Biokinetics Neurotoxicity Repeated exposure 2D mouse brain cell culture 3D rat brain cell culture abstract The difficulty in mimicking nervous system complexity and cell–cell interactions as well as the lack of kinetics information has limited the use of in vitro neurotoxicity data. Here, we assessed the biokinetic profile as well as the neurotoxicity of Amiodarone after acute and repeated exposure in two advanced rodent brain cell culture models, consisting of both neurons and glial cells organized in 2 or 3 dimensions to mimic the brain histiotypic structure and function. A strategy was applied to evidence the abiotic processes possibly affecting Amiodarone in vitro bioavailability, showing its ability to adsorb to the plastic devices. At clinically relevant Amiodarone concentrations, known to induce neurotoxicity in some patients during therapeutic treatment, a complete uptake was observed in both models in 24 h, after single exposure. After repeated treatments, bioaccumulation was observed, especially in the 3D cell model, together with a greater alteration of neurotoxicity markers. After 14 days, Amiodarone major oxidative metabolite (mono-N-desethylamiodarone) was detected at limited levels, indicating the presence of active drug metabolism enzymes (i.e. cytochrome P450) in both models. The assessment of biokinetics provides useful information on the relevance of in vitro toxicity data and should be considered in the design of an Integrated Testing Strategy aimed to identify specific neurotoxic alerts, and to improve the neurotoxicity assay predictivity for human acute and repeated exposure. Ó 2015 Published by Elsevier Ltd. 1. Introduction The nervous system is one of the most complex organ systems in terms of both structure and function; in addition, the lack of regeneration after severe damage renders the nervous system particularly vulnerable to toxic insult (Gramowski et al., 2004). Neurotoxicity is indeed one of the toxicity endpoints generally assessed in the safety evaluation of many chemicals as requested by EU Regulations, such as EC Regulations 1907/2006 (REACH); 1107/2009 (pesticides) and 528/2012 (biocides). Last but not least, the increasing onset of neuronal disorders and neurodegenerative diseases, linked to the aging of the population, represents a clear demand for drugs active on the nervous system for which safety has to be assessed in the early phase of development. The currently accepted neurotoxicity studies include in vivo tests, usually on rodents (Bal-Price et al., 2010a); in this kind of stud- ies direct adverse effects on the nervous system are often difficult to distinguish from indirect effects, linked to hormonal and immuno- logical stimuli. This makes the interpretation of the observed functional changes quite difficult and leads to the conclusion that in vivo toxicity tests are not always ideal for the detection of neurotoxic effects (Harry and Tiffany-Castiglioni, 2005). http://dx.doi.org/10.1016/j.tiv.2015.01.012 0887-2333/Ó 2015 Published by Elsevier Ltd. Abbreviations: AMI, Amiodarone; MDEA, mono-N-desethylamiodarone; DDEA, di-N-desethylamiodarone; 2D mouse model, murine neuronal network culture in two dimensional structure; 3D rat model, re-aggregating rat brain cell cultures in three dimensional structure; DIV, days in vitro; LDH, lactate dehydrogenase; CYP, cytochrome P450; DMSO, Dimethylsulfoxide; DMEM, Dulbecco’s Modified Eagle’s medium; PBS, phosphate buffered saline; PNGM, Primary Neuron Growth Medium; PNBM, Primary Neuron Basal Medium; d0, day 0; d13, day 13; TP, time point; LOD, limit of detection; LOQ, limit of quantification; BBB, blood–brain barrier. ⇑ Corresponding author at: Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. Tel.: +39 (0)6 49902959. E-mail address: emma.diconsiglio@iss.it (E. Di Consiglio). 1 These authors equally contributed to the work. Toxicology in Vitro xxx (2015) xxx–xxx Contents lists available at ScienceDirect Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit Please cite this article in press as: Pomponio, G., et al. Amiodarone biokinetics, the formation of its major oxidative metabolite and neurotoxicity after acute and repeated exposure of brain cell cultures. Toxicol. in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.01.012