Amiodarone biokinetics, the formation of its major oxidative metabolite and neurotoxicity after acute and repeated exposure of brain cell cultures
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 studies direct adverse effects on the nervous system are often difficult to distinguish from indirect effects, linked to hormonal and immunological 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).
There is nowadays a large consensus that the animal testing needs to be replaced by a combination of in silico and in vitro approaches, as evidenced by ethical and economic arguments, besides scientific ones (Bal-Price et al., 2010a). However, so far in vitro studies have been proposed to be used as complementary to animal testing (Harry et al., 1998, Bal-Price et al., 2010b), essentially to support the development of biomarkers as early indicators of adverse effects (e.g. initial biochemical alterations), or the identification of mechanism of action. Indeed, none of the in vitro available tests could be considered as a standing alone method, relying on for the evaluation of the neurotoxic hazard of a chemical. This is mainly related to the complex anatomical structure of the nervous system, its physiology, as well as cell–cell interaction among different cell types (neuronal and glial) (Weiss, 2011). However, promising models to be used as alternatives to in vivo neurotoxicity testing have been proposed (Bal-Price et al., 2010b), addressing the issues of complexity and cell–cell interactions, such as (i) the primary cortical (glia-containing) murine neuronal network culture, established on the surface of micro-electrode arrays (neurochips) in a two dimensional structure (2D mouse model) (Weiss, 2011) and (ii) the tri-dimensional re-aggregating rat brain cell cultures (3D rat model), containing different brain cell types (Honegger and Zurich, 2011). The 2D mouse model allows to directly monitor changes of 200 features of the electrical firing patterns by deriving quantitative parameters belonging to five general categories: burst structure, oscillatory aspects, synchronicity, connectivity and general activity (Gramowski et al., 2010, Johnstone et al., 2010, Weiss, 2011). The 3D rat model is composed of neurons, astrocytes, oligodendrocytes and microglial cells, allowing multiple cell–cell interactions, and the development of histotypic structures such as extracellular matrix, synapses and myelinated axons (Zurich et al., 2004). It has been extensively used for neurotoxicological investigations, and several structural and functional endpoints have proved to be useful specific markers of neurotoxicity, such as the activity of cell type-specific enzymes, the expression of selected genes, as well as astroglial and microglial reactivities (Monnet-Tschudi et al., 1995, Zurich et al., 2004, Zurich et al., 2013).
The long-term stability of both models makes them suitable for both acute and repeated exposure to chemicals or drugs, giving in addition the possibility to study different functional endpoints, allowing to distinguish between general cytotoxicity and possible specific chemical-induced neurotoxicity.
The combination of the 2D and 3D brain cell models with a blood brain barrier (BBB) in vitro model (Culot et al., 2013) was evaluated as an improved in vitro neurotoxicity testing strategy (Schultz et al., 2015), aimed to overcome the lack of the in vivo filtering processes associated with the BBB or lipid-aqueous partitioning, contributing to the artificial nature of the culture exposure (Hallier-Vanuxeem et al., 2009). However, another important step forward to increase the predictability of in vitro testing is the measurement of in vitro kinetics, including all the abiotic and biological processes such as in situ metabolic capability, determining the actual exposure of cells, which can greatly influence the toxicological outcome, especially after repeated exposure (Coecke et al., 2013). Despite its relevance, this aspect is most of the times neglected, and the nominal concentration is used to refer to concentration-effect relationship.
Here we describe the in vitro biokinetics of Amiodarone (2-n-butyl-3-[3,5-diiodo-4-diethylaminoethoxybenzoyl]-benzofuran; AMI), the formation of its major oxidative metabolite mono-N-desethylamiodarone (MDEA), and some neurotoxicity end-points induced after acute and 14 day-repeated treatments in the 2D and 3D brain models. AMI, a Class III antiarrhythmic medication, is a non-competitive inhibitor of alpha- and beta-adrenergic receptors, commonly used to treat patients with refractory ventricular tachycardia and paroxysmal atrial fibrillation. In some patients under therapeutic treatment, during which the drug reaches plasma concentrations in the range of 1.3–2 μM (Lafuente-Lafuente et al., 2009), AMI is known to induce a variety of side effects including neurotoxicity (Brief et al., 1987), characterised by a set of symptoms such as headache, dizziness, fatigue, tremor, peripheral sensorimotor neuropathy, proximal muscle weakness, ataxia, with few cases of parkinsonism (Ishida et al., 2010).
Section snippets
Chemicals and reagents
AMI was purchased from Sigma–Aldrich (Switzerland, Germany and Italy, catalogue No A8423-1g, lot #109K1307), MDEA (purity: 98.9%) was synthesized and kindly provided by Sanofi-Aventis (Germany, catalogue No RDS 8262). Dimethylsulfoxide (DMSO) (purity ⩾99.9%; Sigma–Aldrich catalogue No 472301). For AMI and MDEA quantification HPLC grade chemicals were obtained from commercially available sources. The Milli-Q water purification system (Millipore, Italy) was used to obtain deionised water.
Stability, solubility and cross-contamination among wells of the test compound
Cytotoxicity testing
As shown in Fig. 1, the cell death induced by AMI treatment in the 2D mouse model was characterised by a flat dose–response in the range of the four tested concentrations (0.0035–10 μM), with apparently no differences between single and repeated exposure (although the latter is likely underestimated due to the limit of measuring released LDH over time). After a single treatment, AMI was not cytotoxic at 0.0035 μM, whereas at 0.350 and 1.25 μM AMI about 10–20% cell death was measured (no
Discussion
In vitro testing assessing the toxicity of chemicals and drugs has shown so far a limited capacity of correlation with in vivo effects, especially for neurotoxicants, due to both the complex structure and functionality of the nervous system and the lack of kinetics information.
These two issues were tentatively addressed within the European Seventh Framework Program Project Predict-IV (FP7/2009–2013) in the frame of which the present work was conducted. Biokinetic parameters in relation to
Conflict of Interest
The authors declare that there are no conflicts of interest.
Transparency Document
Acknowledgments
This project was funded by the European Union’s 7th Framework Programme under grant agreement no. 202222, Predict-IV. We thank Hannelore Popa-Henning for the administrative work and organization and Denise Tavel for technical support.
References (41)
- et al.
Relevance of in vitro neurotoxicity testing for regulatory requirements: challenges to be considered
Neurotoxicol. Teratol.
(2010) - et al.
In vitro developmental neurotoxicity (DNT) testing: relevant models and endpoints
Neurotoxicology
(2010) - et al.
Toxicokinetics as a key to the integrated toxicity risk assessment based primarily on non-animal approaches
Toxicol. In Vitro
(2013) - et al.
New strategy for alerting central nervous system toxicity: integration of blood–brain barrier toxicity and permeability in neurotoxicity assessment
Toxicol. In Vitro
(2009) - et al.
Microelectrode arrays: a physiologically based neurotoxicity testing platform for the 21st century
Neurotoxicology
(2010) - et al.
Electrophysiologic effects of desethylamiodarone, an active metabolite of amiodarone: comparison with amiodarone during chronic administration in rabbits
Am. Heart J.
(1988) - et al.
Efflux transport of N-monodesethylamiodarone by the human intestinal cell-line Caco-2 cells
Drug Metab. Pharmacok.
(2007) - et al.
Quantitative determination of glutamate mediated cortical neuronal injury in cell culture by lactate dehydrogenase efflux assay
J. Neurosci. Meth.
(1987) - et al.
Severe ataxia caused by amiodarone
Am. J. Cardiol.
(2005) - et al.
Strategies and tools for preventing neurotoxicity: to test, to predict and how to do it
Neurotoxicology
(2012)
Microglial responsiveness as a sensitive marker for trimethyltin (TMT) neurotoxicity
Brain Res.
In vitro kinetics of amiodarone and its major metabolite in two human liver cell models after acute and repeated treatments
Toxicol. In Vitro
Antiarrhythmic actions of amiodarone: a profile of a paradoxical agent
Am. J. Cardiol.
High-performance liquid chromatographic assay for amiodarone N-deethylation in microsomes of rat liver
J. Chromatogr.
Markers for gene expression in cultured cells from the nervous system
J. Biol. Chem.
Decarboxylation of l-glutamic acid by brain
J. Biol. Chem.
The role of CYP3A4 in amiodarone-associated toxicity on HepG2 cells
Biochem. Pharmacol.
Evaluation of aggregating brain cell cultures for the detection of acute organ-specific toxicity
Toxicol. In Vitro
Amiodarone and its desethyl metabolite: tissue distribution and morphologic changes during long-term therapy
Circulation
Distribution of amiodarone and its metabolite, desethylamiodarone, in human tissues
Can. J. Physiol. Pharmacol.
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These authors equally contributed to the work.