J Bioenerg Biomembr (2016) 48:413–423 DOI 10.1007/s10863-016-9673-9 Role of mitochondrial complex I and protective effect of CoQ10 supplementation in propofol induced cytotoxicity Christian Bergamini1 & Noah Moruzzi2 & Francesco Volta2 & Laura Faccioli1 & Jantje Gerdes2 & Maria Cristina Mondardini3 & Romana Fato1 Received: 16 June 2016 /Accepted: 2 August 2016 /Published online: 15 August 2016 # Springer Science+Business Media New York 2016 Abstract Propofol (2,6-diisopropylphenol) is an anaesthetic of the enzyme. Cells supplemented with CoQ10 are more resis- widely used for human sedation. Due to its intrinsic antioxidant tant to propofol toxicity. Propofol exposure induces cellular properties, rapid induction of anaesthesia and fast recovery, it is damages due to mitochondrial impairment. The site of propofol employed in paediatric anaesthesia and in the intensive care of inhibition on Complex I is the Q module. CoQ10 supplementa- premature infants. Recent studies have pointed out that expo- tion protects cells against the loss of energy suggesting its pos- sure to anaesthesia in the early stage of life might be responsible sible therapeutic role to minimizing the detrimental effects of of long-lasting cognitive impairment. The apoptotic neurode- general anaesthesia. generation induced by general anaesthetics (GA) involves mi- tochondrial impairment due to the inhibition of the OXPHOS machinery. In the present work, we aim to identify the main Keywords Propofol . Complex I . Mitochondria . mitochondrial respiratory chain target of propofol toxicity and Anaesthesia . Oxygen consumption to evaluate the possible protective effect of CoQ10 supplemen- tation. The propofol effect on the mitochondrial functionality Abbreviations was assayed in isolated mitochondria and in two cell lines mPTP Mitochondrial permeability transition pore (HeLa and T67) by measuring oxygen consumption rate. The GA General anaesthetics protective effect of CoQ10 was assessed by measuring cells PRIS Propofol infusion Syndrome viability, NADH-oxidase activity and ATP/ADP ratio in cells CoQ1 2,3-Dimethoxy-5-methyl-6-(3-methyl- treated with propofol. Our results show that propofol reduces 2-butenyl)-1,4-benzoquinone cellular oxygen consumption rate acting mainly on mitochon- DB 2,3-Dimethoxy-5-methyl–6-decyl-1, drial Complex I. The kinetic analysis of Complex I inhibition 4-benzoquinone, oxidized form indicates that propofol interferes with the Q module acting as a DBH2 2,3-Dimethoxy-5-methyl–6-decyl-1, non-competitive inhibitor with higher affinity for the free form 4-benzoquinol, reduced form DCIP 2,6-Dichlorophenolindophenol Electronic supplementary material The online version of this article ETC Electron Transfer Chain (doi:10.1007/s10863-016-9673-9) contains supplementary material, BHM Bovine Heart Mitochondria which is available to authorized users. TMPD N,N,N′,N′-Tetramethyl-p-phenylenediamine * Romana Fato FCCP Carbonyl cyanide 4-(trifluoromethoxy) [email protected] phenylhydrazone FMN Flavin mononucleotide 1 Department of Pharmacy and Biotechnology, University of Bologna, MTT 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl- Via Irnerio 48, Bologna, Italy 2H-tetrazolium bromide 2 Institute for Diabetes and Regeneration Research, Helmholtz TMRE Tetramethylrhodamine ethyl ester Zentrum Munich, Parkring 11, 85478 Garching, Germany ECAR Extracellular acidification rate 3 Department of Paediatric Anaesthesia and Intensive Care, S. OCR Oxygen consumption rate Orsola-Malpighi Hospital, Bologna University, Bologna, Italy OXPHOS Oxidative phosphorylation 414 J Bioenerg Biomembr (2016) 48:413–423 Introduction often leading to different and opposite results (Boscolo et al. 2012). Propofol treatment on rat liver isolated mitochondria Propofol is considered a safe agent for human sedation resulted in impairment of mitochondrial function on several because of its peculiar intrinsic antioxidant capacity, its levels: respiratory chain inhibition, ATPase impairment rapid induction (30–60 s.) and fast recovery of anaesthesia (Marian et al. 1997), direct effect on mPTP (Sztark et al. (4–6 min.)(Sebel and Lowdon 1989; Apfelbaum et al. 1995; Javadov et al. 2000;Twaroskietal.2015)and 1993) Due to these features, propofol has also been fre- uncoupling of the respiratory chain (Branca et al. 1995; quently used in paediatric anaesthetic practice and in in- Sztark et al. 1995; Schenkman and Yan 2000;Wuetal. tensive care of premature infants (Istaphanous and Loepke 2005). Moreover, abnormalities in plasma concentration of 2009). However, emerging animal and human data suggest malonylcarnitine and C5-acylcarnitine in a child treated with that high doses or prolonged exposure to propofol could propofol have been reported. This suggests an inhibitory ef- damage the developing nervous system, raising serious fect of propofol on the carnitine palmityl transferase 1, thus questions regarding the safety of propofol for paediatric leading to an impaired fatty-acid oxidation and ATP produc- anaesthesia (Fredriksson et al. 2007; Cattano et al. 2008; tion (Fodale and La Monaca 2008). Recently, Vanlander et al. Bercker et al. 2009; Istaphanous and Loepke 2009; showed that in liver mitochondria enriched fractions from rat Stratmann 2011; Zou et al. 2013). sedated with propofol, Complex II + III activity, but not the Sporadically, propofol causes the so-called propofol infu- activity of the single complexes, was strongly decreased, sion syndrome (PRIS), characterized by metabolic acidosis pointing out an interference with the Coenzyme Q pool. (Fodale and La Monaca 2008) resembling mitochondrial Moreover, a decreased activity in Complex II + III was detect- cytopathies and acquired carnitine deficiency (Marian et al. ed in skeletal muscle homogenate from one patient which died 1997;Wolfetal.2001). These defects occur first in tissues during the acute phase of PRIS (Vanlander et al. 2015). To with high-energy demand such as the heart and muscles. clarify the effect of propofol on mitochondrial respiratory Notably, the use of propofol is discouraged in patients with chain we used both intact cells and isolated mitochondria. In mitochondrial diseases Driessen et al. (Driessen et al. 2007; particular, we investigated the sites of propofol inhibition on Schwartz and Raghunathan 2009). Consistently, it has been mitochondrial Complex I and proposed an inhibition mecha- proposed that children who develop metabolic acidosis and nism. Since the physiological substrate of mitochondrial myocardial failure after propofol infusion may have a subclini- Complex I is Coenzyme Q10 (CoQ10), we investigate the pos- cal form of mitochondrial disease (Parke et al. 1992). The neu- sible protective role of CoQ10 supplementation on cultured rotoxic effect of general anaesthetics (GA) results in an apopto- cells exposed to propofol. tic loss of neurons and is accompanied by an impairment of synaptic development associated to modification of mitochon- drial morphogenesis and function (Pearn et al. 2012). Materials and methods Mitochondrial functionality and cellular energy content are considered important factors in cells survival; modifications Chemicals of these factors can trigger apoptotic cell death. Apoptotic neurodegeneration and neurobehavioral deficits are mainly All chemicals were purchased form Sigma (St. Louis, MO, observed in animals or humans undergoing general anaesthe- USA) unless stated otherwise. sia by propofol during the period of brain growth known as Bbrain growth spurt^ (a period that occurs from the foetal Cell culture development until to about 2 years after birth for humans and 2 or 3 weeks after birth in mice and rats) (Dobbing and HeLa cells and T67 cells (Lauro et al. 1986) were cultured in Sands 1979). During the brain growth spurt the energy de- DMEM (PAA, DASIT, Milan, Italy) supplemented with mand is extremely high and even a partial reduction of energy 2 mM L-glutamine, 10 % (v/v) foetal bovine serum (FBS), supplies can have a strong impact on neural development. 100 U/mL penicillin and 100 μg/mL streptomycin at 37 °C in Retrospective studies on children exposed to general anaes- humidified atmosphere with 5 % CO2. Culture medium was thesia, as well as the results obtained in new-born animals, changed every 2 days and sub-confluent cells were splitted showed a toxic effect of the anaesthetic on the brain devel- using 0.05 % trypsin–EDTA solution. opment that results in long-lasting cognitive impairment (Jevtovic-Todorovic et al. 2003; Fredriksson et al. 2004; Bovine heart mitochondria isolation Fredriksson et al. 2007; Wilder et al. 2009; Paule et al. 2011; Block et al. 2012; Boscolo et al. 2012). Bovine heart mitochondria (BHM) were isolated according to The mitochondrial impairment after propofol administra- Smith, A.L. (Smith 1967) with modifications to allow large- tion has been investigated on several experimental models scale isolation (Pallotti and Lenaz 2007). Protein content was J Bioenerg Biomembr (2016) 48:413–423 415 measured by the Biuret method of Gornall et al. (Gornall et al. and 0.5 % bovine serum albumin (BSA) pH 7.2. The cell 1949) with addition of 10 % sodium deoxycholate using homogenate was frozen and thawed three times to avoid mi- bovine serum albumin (BSA) as standard. BHM were tochondrial compartmentalization. The reaction buffer stored at −80 °C. BHM were frozen and thawed three consisted of 50–100 μg of cellular homogenate, 25 mM po- times before use to avoid compartmentalization. tassium phosphate, 5 mM MgCl2,pH7.2,30°C.Thereaction started by adding