FOURTH INTERNATIONAL FESTEM SYMPOSIUM
In vitro modulation of heavy metal-induced rat liver mitochondria dysfunction: A comparison of copper and mercury with cadmium

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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 incubated 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 tetramethylphenylenediamine 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 Ca2+ 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.

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. Mitochondrial dysfunction is one of the first consequences of heavy metal cytotoxicity. Nevertheless, mechanism(s) underlying the heavy metal-induced mitochondrial dysfunction are not fully elucidated.

Heavy metal divalent cations like Cd2+, Hg2+ and Cu2+ are accumulated in mitochondria that have, as it is assumed, a Ca2+ uniporter channel for Ca2+ uptake (MCU) driven by the trans-membrane potential (ΔΨmito), which is considered to transport these cations as well [1], [2], [3]. Cd2+, besides its ability to be a Ca2+ agonist, has a high affinity for thiol-groups. Both these characteristic features of Cd2+ are attributable for the induction of the mitochondrial permeability transition (MPT) pore opening by this heavy metal [4], [5], [6], [7], [8], [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 mitochondria, is involved both in physiological and pathological processes, including the induction of cell death of different types [10], [11], [12]. Recently we found that the Cd2+-induced rat liver mitochondria (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 Cd2+-induced mitochondrial membrane permeabilization [13], [14], [15]. The production of reactive oxygen species (ROS) is stimulated by Cd2+ as well, which may contribute to its toxicity and induction of apoptosis or necrosis, and to the stimulation of MPT pore opening [16], [17], [18], [19], [20], [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 Hg2+ or Cu2+ ([10], [20], [22], [23] and references therein).

Despite the renowned fact that Cd2+, Hg2+ and Cu2+ increase the ion permeability of the mitochondrial inner membrane and cause a swelling in energized rat heart mitochondria indicating an uptake of K+ [24], [25], the conduction of the detailed comparative study of the heavy metal-induced mitochondrial membrane permeabilization is not up-to-date, anymore. Originally aiming at a comparison of the effects of Cd2+, Hg2+, and Cu2+ on isolated RLM in the absence of Ca2+, Pi and Mg2+ in the assay media, we found that all these heavy metal ions were produced dose-dependently: (i) enhanced membrane permeabilizaton manifested by mitochondrial swelling and activation of the resting state respiration, (ii) inhibition of the uncoupler-stimulated respiration, and (iii) dissipation of the mitochondrial transmembrane potential. The latter, however, could be shown indirectly [23]. Among these metals, Cu2+ exhibited the maximal stimulatory effect on the resting state respiration and the minimal inhibitory action on the 2,4-dinitrophenol-uncoupled respiration, while Cd2+ promoted the strongest depression of the uncoupled respiration and the highest membrane permeabilization to protons. We concluded that both the mtETC and the membrane permeability were the primary targets of the deleterious influence of the heavy metals. In the present study the action of heavy metal cations, Hg2+ and Cu2+, on isolated RLM function in the presence of different MCU, MPT and mtETC effectors was studied and compared to the corresponding effects of Cd2+.

Section snippets

Chemicals

Most of the reagents were purchased from Sigma–Aldrich Company (St. Luis, MO, USA), including CdCl2, HgCl2, and CuCl2. CsA was from Novartis (Basel, Switzerland). The remaining chemicals were of the highest purity commercially available.

Preparation of rat liver mitochondria

Rat liver mitochondria were prepared by differential centrifugation after homogenization in a mannitol–sucrose medium containing 1 mM EGTA and 0.5% bovine serum albumin (BSA) according to Allhire et al. [26]. Mitochondria were washed twice using a medium without

Heavy metals effects on mitochondrial swelling

We found that ruthenium red (RR) and Ru-360, i.e., selective MCU inhibitors, affected the heavy metal-promoted mitochondrial membrane permeabilization differently, depending on the metal species, its concentration, composition of the medium and types of respiratory substrates used for energization of the mitochondria. In isotonic ionic media containing penetrate anions (KSCN, KNO3 and NH4NO3), the MCU inhibitors partially depressed the Cd2+-induced mitochondrial swelling of the non-energized

Conclusion

In conclusion, this study further challenged the cause/consequence relationships underlying Hg2+- and Cu2+-produced mitochondrial dysfunction. Moreover, the comparison of Cu(II) and Hg(II) effects with those of Cd(II) and Ca(II) conducted in this work revealed the significant similarities and distinctions in mechanism(s) of their action on the mitochondrial function, including clear-cut substrate specificity of many effects of these cations. Importantly, the new data pointing to the critical

Acknowledgements

A grant from the Magnus Ehrnrooth Foundation to N.-E. Saris and a grant from the Russian Foundation for Basic Research (No. 07-04-00722) to E.A. Belyaeva are gratefully acknowledged. Because a part of this work was performed at the Viikki Biocenter 1 of the University of Helsinki (Helsinki, Finland), we acknowledge the University for making available to us its equipment and facilities. Many thanks also to Dr. Vera Teplova (selective electrodes) and Dr. Vadim Glazunov (isotope technique) for

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