Toxicology 243 (2008) 51–58 Available online at www.sciencedirect.com Cytotoxic effects of amitriptyline in human fibroblasts A.M. Moreno-Fern´ andez a,∗,1 , M.D. Cordero a,b,1 , M. de Miguel a , M.D. Delgado-Rufino a , J.A. S´ anchez-Alc´ azar b , P. Navas b a Dpto. Citolog´ ıa e Histolog´ ıa Normal y Patol´ ogica, Facultad de Medicina. Universidad de Sevilla, Avda. S´ anchez-Pizju´ an s/n, 41009 Sevilla, Spain b Centro Andaluz de Biolog´ ıa del Desarrollo, ´ Area de Biolog´ ıa Celular, Universidad Pablo de Olavide, 41013 Sevilla, Spain Received 26 July 2007; received in revised form 19 September 2007; accepted 20 September 2007 Available online 26 September 2007 Abstract Amitriptyline is a tricyclic antidepressant widely used in the treatment of chronic pain. The objective of the present study was to investigate the potential cytotoxic effects of amitriptyline in human fibroblasts primary culture. Human fibroblast cells were cultured from healthy subjects and incubated with 50 M and 100 M amitriptyline. Cell counting was performed to study dose- dependency of toxicity. Lipid peroxidation analysis and western blotting for antioxidants catalase and mitochondrial superoxide dismutase (MnSOD) were carried out in order to evaluate oxidative stress. To investigate mitochondria damage the following determinations were made: cytochrome c, citrate synthase, and mitochondrial membrane potential (Ψ m ). Amitriptyline reduced significantly the number of cultured cells, resulting in a decrease of 45.2%, 65.0% and 94.9% when treated with 20 M, 50 M and 100 M amitriptyline, respectively. This drug enhanced the production of oxidized products during lipid peroxidation, inverting the reduced/oxidized ratio to 25% reduction and 75% oxidation after 24 h of amitriptyline administration. A decreased in catalase protein levels has been also observed. Moreover, amitriptyline treatment induced a significant decrease of cytochrome c, Ψ m , and citrate synthase activity; revealing mitochondrial damage. These findings suggest that amitriptyline has a strong cytotoxic effect in human fibroblasts, decreasing growth rate and mitochondrial activity, and increasing oxidative stress. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Amitriptyline; Human fibroblasts; Cytotoxicity; Oxidative stress; Mitochondrial damage 1. Introduction Tricyclic antidepressants, as amitriptyline, have long been used for therapeutic treatment of several neu- ropathic and inflammatory illnesses like fibromyalgia, chronic fatigue syndrome, migraine, irritable bowel syn- drome and atypical facial pain (Gruber et al., 1996). The activity of efficient antidepressants involves many different neurotransmitter systems and receptors, some ∗ Corresponding author. Tel.: +34 95 4551797; fax: +34 95 4551799. E-mail address: anamf@us.es (A.M. Moreno-Fern´ andez). 1 Co-first authors. blocking serotonergic, noradrenergic, or dopaminergic reuptake and others blocking serotonergic receptor or inhibiting monoamine oxidase (Stahl, 1998). In the case of amitriptyline, the drug acts inhibiting serotonin and noradrenaline uptake in presynaptic nerve ending (Maubach et al., 1999). The administration of amitriptyline to cell cultures has been proven to induce several signs of toxicity. The first approaches to elucidate the potential toxicity of this drug suggested alteration of the cellular permeability and a decreased metabolism as secondary effects of amitriptyline (Corona et al., 1973). More recent reports demonstrated that the toxicity of this drug is due to an increase of oxidative stress, generating high amounts of 0300-483X/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2007.09.021