Journal of Trace Elements in Medicine and Biology 25S (2011) S63–S73 Contents lists available at ScienceDirect Journal of Trace Elements in Medicine and Biology journal homepage: www.elsevier.de/jtemb FOURTH INTERNATIONAL FESTEM SYMPOSIUM In vitro modulation of heavy metal-induced rat liver mitochondria dysfunction: A comparison of copper and mercury with cadmium Elena A. Belyaeva a,b,∗ , Sergey M. Korotkov a , Nils-Erik Saris b a I.M. Sechenov Institute of Evolutionary Physiology and Biochemistry RAS, Thorez pr. 44, 194223 St. Petersburg, Russia b Department of Food and Environment Sciences, Viikki Biocenter 1, University of Helsinki, POB 56, FIN-00014 Helsinki, Finland article info Article history: Received 26 August 2010 Accepted 26 October 2010 Keywords: Copper Mercury Cadmium Rat liver mitochondria Mitochondrial dysfunction mechanisms abstract Cadmium (Cd), mercury (Hg) and copper (Cu) are very toxic environmental pollutants that exert their cytotoxic effects as cations by targeting mitochondria. To further underscore molecular mechanism(s) underlying the heavy metal-induced mitochondrial dysfunction we continued to compare the action of Cd, Hg and Cu using a simple and convenient in vitro model, namely isolated rat liver mitochondria incu- bated in assay media of different ionic contents and energized by respiratory substrates, glutamate plus malate for complex I, succinate plus rotenone for complex II, and ascorbate plus tetramethylphenylene- diamine for complex IV. With the help of various selective electrodes, fluorescent probes, isotope and spectrophotofluorometric techniques, significant differences were found in the modulating action of various substances affecting the activity of these respiratory chain complexes and mitochondrial Ca 2+ uniporter or permeability transition pore effectors on the mitochondrial function disturbed by the heavy metals, including clear-cut substrate specificity of many effects of these cations. Sequence of events manifested in the mitochondrial dysfunction produced by the metals under test was elucidated. © 2010 Elsevier GmbH. All rights reserved. Introduction Mitochondrial dysfunction, as known today, plays a critical role in aging, cell death and different diseases, including hereditary ones. It was found also that mitochondria are target organelles for dangerous environmental pollutants such as heavy metals. Mito- chondrial dysfunction is one of the first consequences of heavy metal cytotoxicity. Nevertheless, mechanism(s) underlying the heavy metal-induced mitochondrial dysfunction are not fully elu- cidated. Abbreviations: MCU, mitochondrial Ca 2+ uniporter;  mito , mitochondrial transmembrane potential; MPT, mitochondrial permeability transition; RLM, rat liver mitochondria; CsA, cyclosporine A; mtETC, mitochondrial electron transport chain; ROS, reactive oxygen species; P i , inorganic phosphate; DNP, 2,4-dinitrophenol; EGTA, [ethylenebis(oxyethylene-nitrilo)]tetraacetic acid; BSA, bovine serum albumin; PN, pyridine nucleotides; TPP + , tetraphenylphospho- nium; Rh123, rhodamine 123; DCFH2, 2 ′ ,7 ′ -dichlorodihydrofluorescein; DCF, 2 ′ ,7 ′ -dichlorofluorescein; RR, ruthenium red; Ru-360, ruthenium-360; Glu, glutamate; Mal, malate; Succ, succinate; Asc, ascorbate; TMPD, tetramethyl-p- phenylenediamine; BKA, bongkrekic acid; cyt c, cytochrome c; DTT, dithiothriitol; vit E, vitamin E; FCCP, carbonylcyanide-p-trifluoromethoxyphenyl hydrazone. ∗ Corresponding author at: I.M. Sechenov Institute of Evolutionary Physiology and Biochemistry RAS, Thorez pr. 44, 194223 St. Petersburg, Russia. Tel.: +7 812 555 8532; fax: +7 812 552 3012. E-mail addresses: alenab61@mail.ru, belyaeva@EB10330.spb.edu (E.A. Belyaeva). Heavy metal divalent cations like Cd 2+ , Hg 2+ and Cu 2+ are accumulated in mitochondria that have, as it is assumed, a Ca 2+ uniporter channel for Ca 2+ uptake (MCU) driven by the trans- membrane potential ( mito ), which is considered to transport these cations as well [1–3]. Cd 2+ , besides its ability to be a Ca 2+ agonist, has a high affinity for thiol-groups. Both these char- acteristic features of Cd 2+ are attributable for the induction of the mitochondrial permeability transition (MPT) pore opening by this heavy metal [4–9]. The MPT pore, a voltage-dependent, non-selective high-conductance inner mitochondrial membrane channel of unknown molecular structure, which allows solutes of up to 1500 Da to pass freely in and out of the mitochon- dria, is involved both in physiological and pathological processes, including the induction of cell death of different types [10–12]. Recently we found that the Cd 2+ -induced rat liver mitochon- dria (RLM) permeabilization was affected not only by the MPT pore-opening inhibitors like cyclosporine A (CsA) but also by the mitochondrial electron transport chain (mtETC) inhibitors in a way that indicates the involvement of mtETC complexes in the MPT pore modulation and in the Cd 2+ -induced mitochondrial mem- brane permeabilization [13–15]. The production of reactive oxygen species (ROS) is stimulated by Cd 2+ as well, which may contribute to its toxicity and induction of apoptosis or necrosis, and to the stimulation of MPT pore opening [16–21]. Likewise, in many cases the disturbance of the mtETC, the MPT pore-opening induction and changes in ROS production were found to be involved in the toxic action of Hg 2+ or Cu 2+ ([10,20,22,23] and references therein). 0946-672X/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.jtemb.2010.10.007