The Journal of Membrane Biology https://doi.org/10.1007/s00232-019-00099-w Efect of Triclosan on the Functioning of Liver Mitochondria and Permeability of Erythrocyte Membranes of Marsh Frog (Pelophylax ridibundus (Pallas, 1771)) Mikhail V. Dubinin1 · Kirill S. Tenkov1 · Anton O. Svinin1 · Victor N. Samartsev1 · Konstantin N. Belosludtsev1,2 Received: 28 June 2019 / Accepted: 24 September 2019 © Springer Science+Business Media, LLC, part of Springer Nature 2019 Abstract The paper examines the efects of the antimicrobial agent triclosan on the functioning of the liver mitochondria of marsh frog (Pelophylax ridibundus (Pallas, 1771)). It was established that triclosan inhibits DNP-stimulated respiration of mito- chondria and decreases respiratory control ratio. In addition, triclosan causes the collapse of the mitochondrial membrane potential on both types of substrates. Such an action of triclosan can be mediated by both a protonophore efect and suppres- sion of the activity of complex II and combined activity of complexes II + III (and, to a lesser degree, the combined activity of complexes I + III) of the mitochondrial respiratory chain. It is shown that high concentrations of triclosan enhance the production of hydrogen peroxide during the oxidation of substrates of the complex I by mitochondria, and decrease it in the case of succinate oxidation. It is found that triclosan is able to induce nonspecifc permeability of the liver mitochondria of these amphibians, as well as the plasma membrane of erythrocytes. The possible mechanisms of triclosan efect on marsh frog liver mitochondria and red blood cells are discussed. Graphic Abstract Keywords Triclosan · Toxicology · Mitochondria · Pelophylax ridibundus · Erythrocytes · Membrane permeability Abbreviations TCS Triclosan CsA Cyclosporine * Mikhail V. Dubinin MPT Mitochondrial permeability transition [email protected] RBC Red blood cells Extended author information available on the last page of the article Vol.:(0123456789)1 3 M. V. Dubinin et al. Introduction shown that the amount of triclosan found in fsh living in water polluted by this agent is signifcantly higher than its Triclosan (5-chloro-2′-(2,4-dichlorophenoxy)phenol) content in water, which indicates a strong bioaccumulation (TCS) is a synthetic antimicrobial agent with a broad of this compound. A potential mechanism for the absorp- spectrum of activity. This drug is used in many personal tion of lipophilic pollutants by aquatic organisms is direct care products, medical equipment, veterinary medicine, absorption from water, mainly through the gills, as well as the textile industry, and the production of plastics. The through the skin and with food. Interestingly, in frogs, the antibacterial efect of triclosan has been well studied and absorption of triclosan by the tissues, as well as the coef- is associated with the suppression of the bacterial enoyl- fcient of bioaccumulation, vary considerably depending acyl carrier protein reductase (ENR), a key enzyme in the on the species and stage of development, with the highest biosynthesis of fatty acids. As a result, the synthesis of cell sensitivity observed in the early stages of development membrane phospholipids and further proliferation of bac- (Wang et al. 2018). It is shown that the accumulation of teria are suppressed (Rubin et al. 1999; Levy et al. 1999). triclosan in aquatic organisms can be accompanied by a Eukaryotic organisms do not have the ENR enzyme, number of disorders: deviations in postembryonic develop- so for a long time, it was believed that this agent is harm- ment, impaired functioning of the thyroid gland and liver, less to humans and animals. However, evidence has been and damage to the reproductive system. At the same time, recently accumulated that triclosan afects many biological it is worth noting that high concentrations of triclosan processes in eukaryotes. It is shown that at the organism increased the survival rate of Bufo americanus tadpoles, level, triclosan has a powerful toxic efect, and its accumu- which, apparently, was due to the antimicrobial properties lation in the body can lead to damage to the endocrine sys- of this agent (Smith and Burgett 2005). tem, disruption of muscle contraction, and stimulation of The molecular mechanisms of the toxic efect of triclosan carcinogenesis (Lee et al. 2014; Huang et al. 2014; Dhillon on the cells of aquatic organisms are not fully established. et al. 2015; Kolšek et al. 2015). At the cellular level, this It can be assumed that the toxic efect of this agent may agent afects the functioning of a number of receptors and be associated with mitochondrial dysfunction and permea- intracellular Ca2+ channels, and also causes oxidative bilization of cell membranes, like in mammals. Therefore, stress and cell death (Cherednichenko et al. 2012; Zorov in this work, we studied in vitro the efect of triclosan on et al. 2014; Kolšek et al. 2015). the parameters of respiration and oxidative phosphorylation It is assumed that the effects of triclosan may be of marsh frog (Pelophylax ridibundus (Pallas, 1771)) liver mediated by its efect on the permeability of biological mitochondria, the activity of the respiratory chain complexes membranes and the functioning of membrane-associated of organelles and the production of hydrogen peroxide. In enzymes. One of the targets of the toxic efect of triclosan addition, we studied the efect of this agent on the perme- is mitochondria, since these organelles are responsible for ability of the inner membrane of organelles, as well as the initiating apoptosis. In particular, today it is known that plasma membrane of red blood cells. The data obtained triclosan causes a decrease in the mitochondrial membrane indicate that triclosan is capable of causing a complex dis- potential and uncoupling of oxidative phosphorylation in ruption of both the functional activity of marsh frog mito- mammalian mitochondria (Popova et al. 2018). It was chondrial respiratory chain at the level of complex II and suggested that this efect of triclosan may be associated coenzyme Q, and also induce nonspecifc permeabilization with both a specifc uncoupling efect and inhibition of the of the inner mitochondrial membrane and plasma membrane respiratory chain complexes (Teplova et al. 2017; Popova of red blood cells. et al. 2018). On the other hand, we have recently shown that this agent is able to induce disruption of the mem- brane structure of lecithin liposomes and the mitochon- drial inner membrane, which leads to its permeabilization. Materials and Methods Induction of mitochondrial permeabilization can quite well explain the proapoptotic efect of these agents, since in Experimental Animals and Chemicals this case, mitochondria swell and proapoptotic protein cytochrome c is released from organelles (Belosludtsev Mature males of the marsh frog (Pelophylax ridibundus (Pal- et al. 2018). las, 1771)) weighing 60–70 g were caught in the Pine Grove Given the widespread use of triclosan in personal care forest park in the south-eastern part of Yoshkar-Ola (Russia) products, the tendency for this agent to accumulate in in July and August of 2018. The study was carried out in aquatic ecosystems and sludge deposits is not surprising accordance with the European Convention for the Protec- (Dhillon et al. 2015; Weatherly and Gosse 2017). It is tion of Vertebrates used for experimental and other purposes (1986) and the principles of the Helsinki Declaration (2000). 1 3 Efect of Triclosan on the Functioning of Liver Mitochondria and Permeability of Erythrocyte… All the experimental protocols were approved by the Mari mL) were subjected to three cycles of freezing/thawing at State University Ethics Committee (Yoshkar-Ola, Russia). − 20/+ 30 °C in a hypotonic bufer, containing 10 mM Tris/ All chemicals were purchased from Sigma-Aldrich HCl, pH 7.6. The composition of the bufers used for the (USA). analysis of activity of individual complexes is given in pro- tocol (Spinazzi et al. 2012). The activity of complex I was Isolation of Mitochondria estimated at 25 °C by the efciency of oxidation of added NADH by the suspension of disrupted mitochondria, which Mitochondria from the liver of animals were isolated by the was followed by the decrease of absorbance at 340 nm. The conventional method of diferential centrifugation (Rous- activity of complex II was evaluated at 37 °C (in order to sel et al. 2015). The isolation medium contained 250 mM fully activate the enzyme) by the efciency of reduction of sucrose, 1 mM EGTA, and 3 mM Tris/HCl buffer (pH 2,6-dicholrophenol indophenol (sodium salt) by the sus- 7.3). The protein content of the mitochondrial prepara- pension of disrupted mitochondria in the presence of suc- tion was assayed at 540 nm using the biuret method with cinate, which was followed by the decrease of absorbance at bovine serum albumin used as a standard. The mitochon- 600 nm. The activity of complex III was measured at 25 °C drial preparation from frog livers contains a dark pigment by the efciency of reduction of added cytochrome c by which absorbs at 540 nm; therefore, the absorbance of the the suspension of disrupted mitochondria, which was fol- same volume of mitochondria in isolation bufer contain- lowed by the decrease of absorbance at 550 nm. The activ- ing 0.6% potassium sodium L(+)-tartrate and 3% NaOH was ity of complex IV was estimated at 25 °C by the efciency subtracted (Roussel et al. 2015). During the experiment, the of oxidation of added cytochrome c (preliminary reduced suspension of mitochondria (20–30 mg of mitochondrial by the suspension of disrupted mitochondria as described protein in 1 mL) was stored on ice in a narrow plastic tube. in protocol (Spinazzi et al. 2012), which was followed by the decrease of absorbance at 550 nm. The activities of the Mitochondrial Respiration and Phosphorylation respiratory chain complexes (in the absence and presence of triclosan) were registered within 2–3 min after the beginning The oxygen consumption rate of mitochondria was moni- of the redox reaction (nmol/min/mg protein).