Modular kinetic analysis reveals differences in Cd2+ and Cu2+ ion-induced impairment of oxidative phosphorylation in liver Jolita Ciapaite1, Zita Nauciene1,2, Rasa Baniene2, Marijke J Wagner3, Klaas Krab3 and Vida Mildaziene1 Centre of Environmental Research, Faculty of Natural Sciences, Vytautas Magnus University, Kaunas, Lithuania Institute for Biomedical Research, Kaunas Medical University, Lithuania Department of Molecular Cell Physiology, Institute for Molecular Cell Biology, VU University, Amsterdam, The Netherlands Keywords cadmium and copper; lipid peroxidation; metabolic control analysis; modular kinetic analysis; oxidative phosphorylation Correspondence J Ciapaite, Centre of Environmental Research, Faculty of Natural Sciences, Vytautas Magnus University, Vileikos 8, LT-44404 Kaunas, Lithuania Fax: +370 37 327916 Tel: +370 37 455193 E-mail: jolita.ciapaite@falw.vu.nl (Received 29 January 2009, revised 18 April 2009, accepted May 2009) doi:10.1111/j.1742-4658.2009.07084.x Impaired mitochondrial function contributes to copper- and cadmiuminduced cellular dysfunction In this study, we used modular kinetic analysis and metabolic control analysis to assess how Cd2+ and Cu2+ ions affect the kinetics and control of oxidative phosphorylation in isolated rat liver mitochondria For the analysis, the system was modularized in two ways: (a) respiratory chain, phosphorylation and proton leak; and (b) coenzyme Q reduction and oxidation, with the membrane potential (Dw) and fraction of reduced coenzyme Q as the connecting intermediate, respectively Modular kinetic analysis results indicate that both Cd2+ and Cu2+ ions inhibited the respiratory chain downstream of coenzyme Q Moreover, Cu2+, but not Cd2+ ions stimulated proton leak kinetics at high Dw values Further analysis showed that this difference can be explained by Cu2+ ion-induced production of reactive oxygen species and membrane lipid peroxidation In agreement with modular kinetic analysis data, metabolic control analysis showed that Cd2+ and Cu2+ ions increased control of the respiratory and phosphorylation flux by the respiratory chain module (mainly because of an increase in the control exerted by cytochrome bc1 and cytochrome c oxidase), decreased control by the phosphorylation module and increased negative control of the phosphorylation flux by the proton leak module In summary, we showed that there is a subtle difference in the mode of action of Cd2+ and Cu2+ ions on the mitochondrial function, which is related to the ability of Cu2+ ions to induce reactive oxygen species production and lipid peroxidation Many pollutants, even at low effective concentrations, can harm living organisms by weakening their ability to cope with long-term environmental challenges At excess amounts, the heavy metals cadmium and copper are toxic and carcinogenic [1] The ability of cadmium and copper to accumulate in the bones, liver and kidneys determines their toxicity Their deleterious effects can be ameliorated to some extent by binding to metallothionein [2] Cellular dysfunction induced by cadmium and copper is thought to involve alterations Abbreviations CiJP , flux control coefficient, quantifying the control of phosphorylation flux JP by module i; CiJR , flux control coefficient, quantifying the control of respiratory flux JR by module i; CoQ, coenzyme Q; COX, cytochrome c oxidase; DCPIP, 2,6-dichlorophenolindophenol; JL, proton leak flux; JP, phosphorylation flux; JR, respiratory flux; MCA, metabolic control analysis; ROS, reactive oxygen species; SDH, succinate dehydrogenase; TBA, 2-thiobarbituric acid; TBARS, thiobarbituric acid reactive substances; XCoQ, fraction of reduced coenzyme Q; Dp, proton-motive force; Dw, mitochondrial transmembrane electric potential 3656 FEBS Journal 276 (2009) 3656–3668 ª 2009 The Authors Journal compilation ª 2009 FEBS J Ciapaite et al in mitochondrial metabolism Initially, the accumulation of metal ions in mitochondria may protect the cell against metal overload, however, later, their incorporation may cause complex disturbances in mitochondrial function [3–5], resulting in severe defects in cellular metabolism Cd2+ ions are transported into the mitochondria via a Ca2+ -uniporter [6], whereas the accumulation of Cu2+ in mitochondria proceeds via a different, energyindependent mechanism [7] Both metal ions interact with important functional groups (in particular, thiol groups) in a variety of enzymes in the matrix and inner mitochondrial membrane [8] At micromolar concentrations, Cd2+ ions uncouple oxidative phosphorylation and inhibit respiration in actively ADP-phosphorylating (state 3) isolated rat liver mitochondria [4] At higher concentrations, Cd2+ ions inhibit succinate dehydrogenase (SDH) and H+-ATPase [5,9] Increasing the amount of Cu2+ ions added per mg of mitochondrial protein has been shown to successively cause inhibition of phosphate transport, accumulation of K+ ions, membrane aggregation, stimulation of respiration in the absence of active ADP phosphorylation (state 4), an increase in passive membrane permeability to cations and anions, uncoupling and swelling, and inhibition of respiration in state [3,4] Furthermore, it has been suggested that Cu2+ ions inhibit SDH [10] Cd2+ and Cu2+ ions induce cell death by necrosis and apoptosis via mechanisms involving opening of the mitochondrial permeability transition pore and increased generation of reactive oxygen species (ROS) [11–13] In turn, metal-induced stimulation of ROS production has been suggested to stem from both increased ROS production by the mitochondrial respiratory chain and decreased activity of the antioxidant enzymes [9,11,14–16] Although the effects of Cd2+ and Cu2+ ions on some individual mitochondrial enzymes and processes have been studied extensively, few attempts have been made to elucidate the mode of action of Cd2+ and Cu2+ ions at the system level, which, in turn, would allow us to understand the complex metabolic effects of these substances Metabolic control analysis (MCA) is a useful tool for studying complex biological systems because it allows the quantification of the contribution made by each system component to system behavior (e.g fluxes, metabolite concentrations) in terms of control coefficients [17,18] In turn, knowledge of the system’s control structure is valuable in that it allows identification of system components that are potentially most important in mediating the effects of external effectors on the system (i.e the component with the highest control Metal-induced impairment of mitochondrial function coefficient) [19] A ‘top-down’ elasticity analysis (or modular kinetic analysis) was developed to simplify experimental assessment of the control structure of the complex system via MCA [20], and was initially used to study the control of fluxes and intermediates in the oxidative phosphorylation system [21] The method is also valuable in determining the sites of action of external effectors within a system [22–27] In this type of analysis, the system of interest is conceptually subdivided into functional modules (reaction blocks) in such a way that the selected modules interact via a single connecting intermediate In the further analysis, each module is treated as a single enzyme Figure 1A shows how the oxidative phosphorylation system can be subdivided into three functional modules (respiratory chain, phosphorylating and proton leak module) with membrane Fig Modularization of the system Division of the oxidative phosphorylation system into (A) the respiratory chain, phosphorylation and proton leak modules, with Dw as the connecting intermediate; (B) the CoQ-reducing and CoQ-oxidizing modules with fraction of reduced CoQ (XCoQ) as the connecting intermediate; and (C) the CoQ-reducing, cytochrome bc1 + COX, phosphorylation and proton leak modules with Dw and XCoQ as the connecting intermediates Succ, succinate; SDH, dicarboxylate carrier and succinate dehydrogenase; cyt bc1, cytochrome bc1, COX, cytochrome c oxidase, XCoQ, fraction of reduced CoQ Arrows marked e and h indicate electron and trans-membrane proton flux, respectively (in this study all fluxes were analyzed in terms of oxygen consumption flux) FEBS Journal 276 (2009) 3656–3668 ª 2009 The Authors Journal compilation ª 2009 FEBS 3657 Metal-induced impairment of mitochondrial function J Ciapaite et al potential (Dw) as the connecting intermediate [21] To obtain module kinetics, titrations with module-specific inhibitors are performed and the flux and level of the connecting intermediate measured Elasticity coefficients, quantifying sensitivity of the flux through the module to a change in the level of the connecting intermediate, can be calculated from the slope of the inhibitor titration curves at a steady state In turn, elasticity coefficients and steady-state flux values can be used to calculate the flux control coefficients of the modules [21] Repeating the procedure in the presence of a fixed concentration of external effector reveals how that effector affects the kinetics of each module and the magnitude of the control that each module exerts over system fluxes The drawback of the ‘top-down’ approach to MCA is that it yields a coarse picture of the control structure of the system Different ways of modularizing the system of interest may allow a more resolved picture However, this is often limited by the feasibility of assigning modules that interact via a single connecting intermediate [28] In this study, we used modular kinetic analysis and MCA to determine the effects of low concentrations (5 lm) of CdCl2 and CuCl2 on the kinetics and control of oxidative phosphorylation in isolated rat liver mitochondria respiring on succinate To obtain a more resolved picture of the effects of Cd2+ and Cu2+ ions on the system, we subdivided the oxidative phosphorylation system into modules in different ways (Fig 1) We showed that at the concentration tested, both metal ions inhibited respiratory chain module components downstream of coenzyme Q (CoQ) In addition, Cu2+ ions increased the permeability of the inner membrane to ions at high Dw levels We tested a hypothesis that the latter effect resulted from Cu2+ ion-induced formation of ROS and lipid peroxidation Results ence of Cd2+ ions than in their absence, when compared at the same Dw value (Fig 2B) The kinetics of the proton leak and phosphorylation modules were not significantly affected by Cd2+ ions (Fig 2A,C), as indicated by similar values for the proton leak (JL) and phosphorylation (JP) flux in the presence and absence of CdCl2, when the fluxes are compared at the same Dw value Inhibition of the respiratory chain module by Cd2+ ions resulted in a decrease in Dw by mV in state (i.e the state of maximal ADP phosphorylation) (Table 1) Furthermore, Cd2+ ions decreased JR and JP by 23 and 25%, respectively However, Cd2+ ions had no significant effect on JL in state Three-modular kinetic analysis of the effects of Cu2+ ions is shown in Fig Similar to Cd2+, Cu2+ ions inhibited the respiratory chain module (Fig 3B), although to a lesser extent Cu2+ ions had no significant effect on the kinetics of the phosphorylation module (Fig 3C), but clearly stimulated proton leak kinetics (Fig 3A) The increase in JL was more prominent at higher Dw values corresponding to state (i.e the state with no ADP phosphorylation) (Fig 3A) In state 4, Cu2+ ions stimulated JL by 42% and caused a decrease of 11 mV in Dw In state 3, Cu2+ ions inhibited JR by 16% and JP by 17%, respectively, but had no significant effect on JL (Table 1) Despite moderate effects on the fluxes, in state Cu2+ ions had a strong effect on Dw, which decreased by 12 mV (Table 1) It should be noted that the effects of Cd2+ and Cu2+ ions were determined in two separate series of experiments (performed in spring and autumn, respectively) resulting in two sets of flux and Dw values under control conditions (K1 and K2; Table 1) The difference between the two data sets may have been caused by hormone-related seasonal variations in mitochondrial properties (e.g the expression levels of the enzymes involved in the process of oxidative phosphorylation), as observed in different tissues in rodents [29,30] Three-modular kinetic analysis of effects of Cd2+ and Cu2+ ions on oxidative phosphorylation Bimodulular kinetic analysis of the effects of Cd2+ and Cu2+ ions on oxidative phosphorylation To determine which oxidative phosphorylation components were affected by Cd2+ and Cu2+ ions in liver mitochondria oxidizing succinate, we first used threemodular kinetic analysis with Dw as the connecting intermediate (Fig 1A) We assessed the effects of a low metal ion concentration, which did not induce mitochondrial swelling (results not shown) Figure shows the effect of lm CdCl2 on the kinetics of the three modules The plots indicate that Cd2+ ions inhibited the respiratory chain module because the respiratory flux (JR) is lower in the pres- The data in Figs and indicate that Cd2+ and Cu2+ ions affected the respiratory chain module Therefore, as a next step, we set out to pinpoint the components of the respiratory chain module affected by Cd2+ and Cu2+ ions To achieve this, we conceptually subdivided the oxidative phosphorylation system into two modules: (a) CoQ reducing, comprising dicarboxylate carrier, fumarase and succinate dehydrogenase; and (b) CoQ oxidizing, comprising cytochrome bc1, cytochrome c oxidase (COX) and the rest of the oxidative phosphorylation system, including proton leak and 3658 FEBS Journal 276 (2009) 3656–3668 ª 2009 The Authors Journal compilation ª 2009 FEBS J Ciapaite et al Metal-induced impairment of mitochondrial function JR (nmol O·min–1·mg protein–1) JL (nmol O·min–1·mg protein–1) A B State 150 150 State 50 120 140 160 Δψ (mV) State 100 50 State 100 180 C 150 100 100 100 200 JP (nmol O·min–1·mg protein–1) 200 200 120 140 160 Δψ (mV) 180 50 100 120 140 160 Δψ (mV) 180 Fig Effect of Cd2+ ions on the kinetics of the proton leak module (A), the respiratory chain module (B) and the phosphorylation module (C) The kinetics of the proton leak module were obtained by titrating with a specific inhibitor of the respiratory chain module, malonate (0–12.5 lM), when phosphorylation module activity is fully blocked with oligomycin (0.7 lgỈmL)1) The kinetics of the respiratory chain module were obtained by titrating with a specific inhibitor of the phosphorylation module, carboxyatractyloside (0–0.5 lM) The kinetics of the phosphorylation module were obtained by titrating with a specific inhibitor of the respiratory chain module, malonate (0–3.125 lM), and subsequently calculating JP by subtracting JL from JR at the same value of Dw [21] JR, respiratory flux; JP, phosphorylation flux; JL, proton leak flux Open symbols, no CdCl2 added; closed symbols, plus lM CdCl2 Average of n = independent experiments ± SEM Table Effect of Cd2+ and Cu2+ ions on system properties in state Average of n = independent experiments ± SEM K1 and K2, control experiments with no CdCl2 or CuCl2 added; JR, respiratory flux; JP, phosphorylation flux; JL, proton leak flux K1 JR (nmol min)1 Æmg protein)1) JP (nmol OÆmin)1 Æmg protein)1) JL (nmol OÆmin)1 Æmg protein)1) Dw (mV) lM CdCl2 K2 lM CuCl2 171 ± 14 131 ± 16* 140 ± 118 ± 1* 162 ± 14 122 ± 15* 131 ± 109 ± 2* 8±1 9±2 143 ± 137 ± 2* 9±1 140 ± 10 ± 128 ± 1* *P < 0.05 versus the condition with no CdCl2 or CuCl2 added enzymes involved in ATP synthesis We used the fraction of CoQ (XCoQ) as the connecting intermediate (Fig 1B) The results of bimodular kinetic analysis with XCoQ as the connecting intermediate are presented in Fig A similar JR value at any given XCoQ indicates that the kinetics of the CoQ-reducing module was not significantly affected by either Cd2+ (Fig 4B) or Cu2+ ions (Fig 4D) Both metal ions inhibited the CoQ-oxidazing module (Fig 4A,C) because lower JR values were observed when comparison was made at the same XCoQ level Because three-modular analysis showed that neither of the ions had any effect on the enzymes involved in ATP synthesis (Figs 2C and 3C) or on the proton leak kinetics close to state (Figs 2A and 3A), we can conclude that the site of action of Cd2+ and Cu2+ ions must be cytochrome bc1 and ⁄ or COX Effects of Cd2+ and Cu2+ ions on the activity of succinate dehydrogenase Bimodular kinetic analysis showed that SDH (a component of the CoQ-reducing module) was not significantly affected by either Cd2+ or Cu2+ ions However, literature reports suggest that SDH is the target of both metal ions [5,9,10] To check whether data obtained using modular kinetic analysis were correct, we determined the effect of Cd2+ and Cu2+ ions on SDH activity in isolated rat liver mitochondria The dependence of SDH activity on the concentration of CdCl2 and CuCl2 is shown in Fig 5A and B, respectively At lm neither CdCl2 nor CuCl2 had any significant effect on SDH activity, which was 51 ± nmol 2,6-dichlorophenolindophenol (DCPIP)Ỉ min)1Ỉmg protein)1 under control conditions and 52 ± and 48 ± nmol DCPIPỈmin)1Ỉmg protein)1 in the presence of lm CdCl2 and lm CuCl2, respectively A significant effect on SDH activity was observed only at CdCl2 and CuCl2 concentrations exceeding 10 lm (Fig 5) Effects of Cd2+ and Cu2+ ions on H2O2 production and lipid peroxidation We hypothesized that stronger stimulation of proton leak kinetics by Cu2+ ions (Fig 3A) compared with Cd2+ ions (Fig 2A) may be explained by the ability of Cu2+ ions to stimulate ROS production and induce peroxidation of the membrane lipids Therefore, we FEBS Journal 276 (2009) 3656–3668 ª 2009 The Authors Journal compilation ª 2009 FEBS 3659 Metal-induced impairment of mitochondrial function A 150 B State 150 100 50 120 140 160 Δψ (mV) State 100 50 State 100 180 C 150 100 State 100 200 JP (nmol O·min–1·mg protein–1) 200 JR (nmol O·min–1·mg protein–1) JL (nmol O·min–1·mg protein–1) 200 J Ciapaite et al 120 140 160 Δψ (mV) 180 50 100 120 140 160 Δψ (mV) 180 Fig Effect of Cu2+ ions on the kinetics of the proton leak module (A), the respiratory chain module (B) and the phosphorylation module (C) The kinetics of the modules were obtained as described in the legend for Fig JR, respiratory flux; JP, phosphorylation flux; JL, proton leak flux Open symbols, no CuCl2 added; closed symbols, plus lM CuCl2 Average of n = independent experiments ± SEM 200 State 150 200 State 100 50 20 30 40 50 60 70 80 Fraction of reduced CoQ (%) 250 JR (nmol O·min–1·mg protein–1) B 150 100 50 20 30 40 50 60 70 80 Fraction of reduced CoQ (%) 250 C 200 JR (nmol O·min–1·mg protein–1) 250 A State 150 100 50 0 20 40 60 80 100 Fraction of reduced CoQ (%) JR (nmol O·min–1·mg protein–1) JR (nmol O·min–1·mg protein–1) 250 D 200 State 150 100 50 0 20 40 60 80 100 Fraction of reduced CoQ (%) assessed how Cd2+ and Cu2+ ions affect overall ROS production in isolated mitochondria oxidizing succinate in state (i.e the resting state with no ADP phosphorylation) Figure 6A shows that lm CdCl2 (i.e the concentration used for modular kinetic analysis) had no significant effect on overall H2O2 production, as indicated by the unchanged oxidation rate of 2¢,7¢-dichlorofluorescin (DCF) In turn, lm CuCl2 stimulated the 3660 Fig Effect of Cd2+ and Cu2+ ions on the kinetics of the CoQ-reducing and CoQoxidizing modules Effect of Cd2+ on the kinetics of the CoQ-oxidizing module (A) and CoQ-reducing module (B) Effect of Cu2+ on the kinetics of the CoQ-oxidizing module (C) and CoQ-reducing module (D) The kinetics of the CoQ-oxidizing module were obtained by titrating with a specific inhibitor of the CoQ-reducing module, malonate (0–3.125 lM) The kinetics of the CoQreducing module were obtained by titrating with a specific inhibitor of the CoQ-oxidizing module, myxothiazol (0–80 nM) JR, respiratory flux Open symbols, no CdCl2 or CuCl2 added; closed symbols, plus lM CdCl2 (A,B) or lM CuCl2 (C,D) Average of n = (A,B) and n = (C,D) independent experiments ± SEM rate of DCF oxidation by 43% (Fig 6A) Increasing the concentration of CdCl2 and CuCl2 to 10 lm resulted in DCF oxidation rates that were 1.7 (P < 0.01) and 2.1 (P < 0.01) times higher, respectively Next, we assessed the ability of both metal ions to induce lipid peroxidation in isolated rat liver mitochondria respiring on succinate in state Figure 6B shows the effects of Cd2+ and Cu2+ ions on the formation of FEBS Journal 276 (2009) 3656–3668 ª 2009 The Authors Journal compilation ª 2009 FEBS Metal-induced impairment of mitochondrial function A A 100 80 60 40 20 0 20 40 60 80 CdCl2 (µM) 100 120 SDH activity (% of control) 120 B B 100 80 60 40 20 2+ 20 40 60 80 100 120 140 CuCl2 (µM) 2+ Fig Effect of Cd (A) and Cu (B) ions on succinate dehydrogenase (SDH) activity SDH activity under control conditions (i.e without CdCl2 or CuCl2) was 50.8 ± 7.8 and 50.8 ± 8.3 nmol DCPIPỈ min)1Ỉmg protein)1 (set to 100%) in the experiments shown in (A) and (B), respectively Average of n = independent experiments ± SEM thiobarbituric acid reactive substances (TBARS), which indicate levels of the lipid peroxidation product malondialdehyde At the amounts tested (5 and 10 nmol Cd2+ỈmgỈmitochondrial protein)1), Cd2+ ions had no significant effect on TBARS formation However, for Cu2+ ions, addition of nmolỈmg protein)1 significantly increased (by 26%) the amount of TBARS per mg of mitochondrial protein Increasing the amount of added Cu2+ ions to 10 nmolỈmg protein)1 did not further increase the amount of TBARS formed (Fig 6B) Effects of Cd2+ and Cu2+ ions on the control of fluxes in oxidative phosphorylation Using MCA, we assessed the contribution made by each oxidative phosphorylation module to the control of JR and JP (Table and Fig 7) Three-modular analysis (Fig 1A) revealed that control of JR and JP was shared between the respiratory chain and phosphorylation modules, with the former exerting somewhat more control As expected for state conditions, the proton leak module exerted low levels of positive ** 1000 ** * 500 CuCl2 nmol·mg protein–1 nmol·mg protein–1 10 nmol·mg protein–1 0.15 * * 0.1 0.05 0 μM μM 10 μM 1500 CdCl2 TBARS (nmol·mg protein–1) SDH activity (% of control) 120 2',7'-dichlorofluorescin oxidation rate (RFU·min–1·mg protein–1) J Ciapaite et al CdCl2 CuCl2 Fig Effect of Cd2+ and Cu2+ ions on H2O2 production (A) and lipid peroxidation (B) Average of n = (A) and n = (B) independent experiments ± SEM *P < 0.05 and **P < 0.01 versus the condition with no CdCl2 or CuCl2 added, respectively RFU, relative fluorescence units; TBARS, thiobarbituric acid reactive substances control over JR and low levels of negative control over JP (the latter is because stimulation of this module decreases flux through the phosphorylation branch of oxidative phosphorylation) A somewhat different fluxcontrol pattern was obtained from two separate sets of experiments performed in the absence of CdCl2 and CuCl2 (K1 and K2; Table 2) It has previously been shown that the flux-control structure of the oxidative phosphorylation system is influenced by hormones [31] Therefore, the observed difference may have been caused by seasonal variations in the hormonal state of the animals Bimodular analysis (Fig 1B) showed that the CoQ-reducing module (comprising enzymes involved in substrate transport and SDH) exerted relatively low levels of control over JR and JP compared with the control exerted by the remaining system components Combining the results of three- and bimodular MCA made it possible to deduce the control of fluxes exerted by the respiratory chain complexes downstream of the CoQ (i.e cytochrome bc1 and COX) (Fig 1C) The data obtained showed that cytochrome bc1 and COX together exerted stronger control over fluxes than the components of the respiratory chain upstream of CoQ (Table 2) FEBS Journal 276 (2009) 3656–3668 ª 2009 The Authors Journal compilation ª 2009 FEBS 3661 Metal-induced impairment of mitochondrial function J Ciapaite et al Table Effect of Cd2+ and Cu2+ ions on the metabolic control of fluxes Average of n = independent experiments ± SEM K1 and K2, control experiments with no CdCl2 or CuCl2 added; C, flux control coefficient; JR, respiratory flux; JP, phosphorylation flux CiJR K1 Module, i Respiratory Phosphorylation Proton leak CoQ-oxidizing CoQ-reducing Cytochrome bc1 + COX 0.66 0.33 0.02 0.84 0.16 0.50 lM CdCl2 ± ± ± ± ± ± 0.04 0.04 0.00 0.03 0.03 0.07 K2 0.78 0.20 0.02 0.78 0.22 0.56 ± ± ± ± ± ± 0.02* 0.02* 0.00 0.04 0.04 0.05 0.53 0.45 0.03 0.84 0.16 0.37 lM CuCl2 ± ± ± ± ± ± 0.06 0.06 0.00 0.01 0.01 0.05 0.94 0.06 0.01 0.89 0.11 0.83 ± ± ± ± ± ± 0.02* 0.02* 0.00* 0.00* 0.00* 0.02* ± ± ± ± ± ± 0.02* 0.02* 0.01* 0.00 0.00 0.01* CiJP Module, i K1 Respiratory Phosphorylation Proton leak CoQ-oxidizing CoQ-reducing Cytochrome bc1 + COX 0.68 0.36 )0.03 0.84 0.16 0.51 lM CdCl2 ± ± ± ± ± ± 0.05 0.04 0.01 0.03 0.03 0.08 K2 0.82 0.24 )0.06 0.82 0.18 0.64 ± ± ± ± ± ± 0.02* 0.02* 0.01 0.01 0.01 0.02 0.54 0.49 )0.03 0.88 0.12 0.43 lM CuCl2 ± ± ± ± ± ± 0.07 0.06 0.00 0.01 0.01 0.08 1.00 0.08 )0.08 0.89 0.11 0.89 *P < 0.05 versus condition with no CdCl2 or CuCl2 added Control coefficient of JR A 0.8 0.6 0.4 0.2 Control coefficient of JP CdCl2 K2 CuCl2 K1 B K1 CdCl2 K2 CoQ reduction bc1 + COX Phosphorylation Proton leak CuCl2 0.8 0.6 0.4 0.2 Fig Control distribution of the respiratory (A) and phosphorylation flux (B) among the CoQ-reducing, cytochrome bc1 + COX, phosphorylation and proton leak modules Division of the system of oxidative phosphorylation into modules is depicted schematically in Fig 1C Average of n = data sets JR, respiratory flux; JP, phosphorylation flux; K1 and K2, control experiments with no CdCl2 or CuCl2 added Cd2+ ions induced a redistribution in the control over fluxes through the system (Table and Fig 7) Cd2+ ions tended to increase the control over JR exerted by 3662 the respiratory chain module and significantly decreased the control over JR exerted by the phosphorylation module A similar trend was observed for control over JP; control by the respiratory chain module increased significantly, whereas control by the phosphorylating module decreased significantly In addition, negative control of JP by the proton leak module increased slightly, but significantly Analysis of the control exerted by the respiratory chain components revealed that control of JR and JP exerted by the CoQ-reducing module was not affected by Cd2+ ions Meanwhile, control of JR and JP by cytochrome bc1 and COX tended to increase in the presence of Cd2+ ions The effects of Cu2+ ions on the control pattern followed the same trend as the effects of Cd2+ ions but were more pronounced (Table and Fig 7) Threemodular MCA showed that in the presence of Cu2+ ions the respiratory chain module acquired almost complete control of JR and JP Combination of threeand bimodular analysis revealed that this resulted from a dramatic increase in the control of fluxes by cytochrome bc1 and COX Moreover, in agreement with the observation that Cu2+ ions are more potent stimulators of proton leak kinetics, we showed that the effect of Cu2+ ions on the control of fluxes by the proton leak module was stronger than the effect of Cd2+ ions Taken together, the changes in flux control distribution were consistent with the results of modular kinetic analysis, which revealed that both Cd2+ and Cd2+ ions interfere with oxidative phosphorylation function- FEBS Journal 276 (2009) 3656–3668 ª 2009 The Authors Journal compilation ª 2009 FEBS J Ciapaite et al ing by inhibiting the respiratory chain downstream of CoQ (i.e cytochrome bc1 and ⁄ or COX) Discussion Living systems are continuously exposed to low levels of multi-component pollution, which may affect many cellular processes simultaneously Although no cellular process is hampered severely, there may be a cumulative effect on the functioning of various metabolic pathways and this may ultimately challenge cellular metabolism as a whole Many pollutants, including the heavy metal ions Cd2+ and Cu2+ are expected to interfere with several enzymes at the same time because of a rather nonspecific interaction with their functional groups In this study, we used modular kinetic analysis and MCA to elucidate the molecular mechanisms underlying Cd2+ and Cu2+ ion-induced impairment of the main aerobic energy production pathway in the cell, i.e oxidative phosphorylation By subdividing oxidative phosphorylation into modules in different ways we were able to obtain a detailed picture of the effects of Cd2+ and Cu2+ ions We showed that Cd2+ ions interfere with oxidative phosphorylation solely through their inhibitory effect on respiratory chain complexes downstream of CoQ This resulted in lower respiratory and phosphorylation fluxes and lower Dw values, as well as an increase in flux control by the respiratory chain module The overall effect of Cu2+ ions on oxidative phosphorylation functioning was similar to that seen with Cd2+ ions, however, it was caused not only by inhibition of respiratory chain, but also by stimulation of proton leak module activity at high Dw The latter effect was caused, at least in part, by stimulation of ROS production and the subsequent peroxidation of membrane lipids Modular kinetic analysis uses natural properties of metabolism, i.e its organization into recognizable functional units, simplifying the analysis and making cellular complexity manageable [20,32,33] We demonstrated how different modularization of the system of interest and subsequent application of modular kinetic analysis allows identification of molecular targets of Cd2+ and Cu2+ ions In the first application of the analysis, we conceptually divided oxidative phosphorylation into three functional modules based on the production and consumption of Dw (Fig 1A) We showed that Cd2+ and Cu2+ ions inhibited the respiratory chain module (Figs 2B and 3B) but had no significant effect on the phosphorylation module (Figs 2C and 3C) Interestingly, Cu2+ ions had a stronger ability to uncouple oxidative phosphorylation than did Cd2+ ions, as indicated by stimulation of proton leak module kinetics at high Metal-induced impairment of mitochondrial function Dw values (Figs 2A and 3A) Our data are in agreement with earlier findings that Cu2+ ions can effectively increase membrane permeability, whereas Cd2+ ions are much less effective; comparable swelling of isolated rat liver mitochondria was obtained at lm Cu2+ and 40 lm Cd2+ [34] It has been shown that the ability of Cd2+ ions to increase membrane permeability is dependent on inorganic phosphate transport and increases with increasing phosphate concentration in the medium [35] Therefore, under certain experimental conditions characterized by a high inorganic phosphate concentration, low Cd2+ concentrations can induce uncoupling [35,36] However, at a low inorganic phosphate concentration (as in this study) a much higher Cd2+ concentration is needed to stimulate membrane ion permeability and induce uncoupling [37] Three-modular kinetic analysis has been used previously to localize the sites of action of Cd2+ ions in isolated potato tuber mitochondria respiring on succinate, and has shown that different concentrations of Cd2+ ions inhibited the respiratory chain module, had no significant effect on the phosphorylation module and stimulated proton leak kinetics at 3.5 lm free Cd2+ [22] The apparent contradiction between the latter finding and our observation that lm CdCl2 has no significant effect on the proton leak kinetics is explained by the fact that we used a lower amount of Cd2+ ions per mg of mitochondrial protein, and therefore uncoupling did not occur After establishing that the respiratory chain module is the target of both metal ions, we conceptually divided the system into CoQ-reducing and CoQ-oxidizing modules with XCoQ as the connecting intermediate (Fig 1B) This approach revealed that lm CdCl2 or CuCl2 interfered with the respiratory chain complexes downstream of CoQ and had no significant effect on the dicarboxylate carrier and SDH (Fig 4) By contrast, earlier studies identified SDH as the target of both metal ions [5,9,10] We investigated this and found that lm CdCl2 or CuCl2 did not affect SDH activity under our experimental conditions (Fig 5), confirming that modular kinetic analysis yielded the correct results In this study, we showed that at low concentrations, Cd2+ and Cu2+ ions affected a rather limited number of components in the oxidative phosphorylation system, i.e the respiratory chain downstream of CoQ (i.e cytochrome bc1 and COX) and the mitochondrial inner membrane This suggests that because these components are affected by a low concentration of Cd2+ and Cu2+ ions, they are the most sensitive and are therefore responsible for the early response of the system when it is chronically exposed to low levels of pollutants Support for this hypothesis comes from the observation that mitochon- FEBS Journal 276 (2009) 3656–3668 ª 2009 The Authors Journal compilation ª 2009 FEBS 3663 Metal-induced impairment of mitochondrial function J Ciapaite et al dria from liver, kidney and muscle of cadmium-treated rats display a rapid early decrease in COX activity, followed by partial restoration after months of treatment, and a progressive decrease in SDH activity [9] The variation in the number of molecular targets of Cd2+ or Cu2+ ions reported in the literature may be explained by their differing sensitivity to these ions, resulting in a greater number of affected system components at higher ion concentrations For example, the effects on mitochondrial ATPase were examined at Cd2+ concentrations in the mm range (up to 10 mm) [5] It is questionable whether such high concentrations of Cd2+ ions can be achieved even in a heavily intoxicated cell Furthermore, it has been shown that ATPase activity was induced by very low concentrations of Cu2+ (4–6 nmolỈmg protein)1) However, the same ion concentration induced maximal uncoupling under the experimental conditions used by Hwang et al [34], and the effect might therefore be explained by uncoupling In contrast to these findings, our data (Figs 2C and 3C) did not reveal any noticeable effects of lm CdCl2 and lm CuCl2 on the components of the ADP phosphorylation machinery We hypothesized that the different effect of Cd2+ and Cu2+ ions on the proton leak kinetics observed in our study may be determined by the ability of Cu2+ ions to stimulate ROS production, which in turn may lead to lipid peroxidation and membrane damage We showed that lm CuCl2, but not lm CdCl2, stimulated the oxidation of DCF significantly, indicating increased H2O2 formation in the mitochondrial matrix Because oxygen favors reduction by one electron at a time, the primary ROS that is formed by the action of Cu2+ ions must be a superoxide anion radical The latter may reduce Cu2+ to Cu+, leading to formation of the hydroxyl radical via the Haber–Weiss cycle [38] In turn, the hydroxyl radical is a potent inductor of lipid peroxidation In support of this, we showed that Cu2+ ions, but not Cd2+, cause accumulation of TBARS, suggesting that Cu2+ may increase membrane permeability by stimulating lipid peroxidation It has also been shown that, in intact hepatocytes, Cu2+ ions are much more potent inductors of lipid peroxidation than Cd2+ ions [14] In addition to the evaluation of Cd2+ and Cu2+ ioninduced changes in the kinetics of the individual oxidative phosphorylation components, we were also interested in how these changes affect the control structure of the system MCA was designed to deal with the effects of a small disturbance in enzyme activity on system fluxes and is a useful method with which to analyze and diagnose cell sickliness caused by agents that simultaneously affect many enzymes In this study, we assessed metabolic control of the respiratory (JR) and 3664 phosphorylation (JP) fluxes by the respiratory chain, phosphorylation and proton leak modules, as well as control of the respiratory flux by CoQ-reducing and CoQ-oxidizing modules, and determined how Cd2+ and Cu2+ ions affected this control In agreement with previously published data [21,24], we found that control of both fluxes is mainly shared between the respiratory chain and phosphorylation modules, with only slight control being exerted by the proton leak module (positive in the case of JR and negative in the case of JP) (Table and Fig 7) Five micromolar CdCl2 and CuCl2 caused an increase in the control of JR and JP by the respiratory chain module, with cytochrome bc1 and COX being the main contributors This is in agreement with our observation that both metal ions interfere with the respiratory chain components downstream of CoQ The effect of Cu2+ ions was stronger than that of Cd2+ Furthermore, Cu2+ ions increased negative control of JP by the proton leak module more than Cd2+ ions, because the former had a stronger effect on the permeability of the inner membrane to ions This observation illustrates how interfering with a system component that makes a relatively low contribution to the control of system fluxes (i.e proton leak) may compromise the system via control of other vital system properties (e.g stronger membrane damage by Cu2+ ions may contribute to inhibition of cytochrome bc1 and COX, which are membrane proteins, leading to increased flux control by these enzymes) Flux control is a property of the whole system rather than of individual components of that system (i.e enzymes, modules), as illustrated by the summation theorem for flux control coefficients [17,18] As a result, individual flux control coefficients for each system component cannot change independently Consequently, we see that an increase in flux control by the respiratory chain and proton leak modules results in decreased control of both fluxes by the phosphorylation module, although this module is not affected directly by Cd2+ and Cu2+ ions Taken together, we have demonstrated how modular kinetic analysis could be gradually applied to identify the sites of action of external effectors such as Cd2+ and Cu2+ ions in a multienzyme system like oxidative phosphorylation Although we found that Cd2+ ions affect system behavior by acting on a single target, whereas Cu2+ interferes with two, there is no doubt that this method is valuable, especially when assessing multisite effects of toxic substances on complex metabolic systems The next step in this type of analysis may be to elucidate the combined effects of several effectors (e.g a mixture of Cd2+ and Cu2+ ions) with individual known sites of action Repeated modular FEBS Journal 276 (2009) 3656–3668 ª 2009 The Authors Journal compilation ª 2009 FEBS J Ciapaite et al kinetic analysis using different modularization approaches might reveal the causes and consequences of their competition for binding to the same molecular component or synergism of their action Materials and methods Materials Rotenone, myxothiazol, oligomycin, creatine phosphokinase, CoQ1, 2¢,7¢-DCF, 2¢,7¢-dichlorofluorescin diacetate and 2-thiobarbituric acid (TBA) were from Sigma (SigmaAldrich, St Louis, MO, USA) Isolation of mitochondria The handling of the animals conformed to the rules defined by the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (License No 0006 of State Veterinary Service for working with laboratory animals) Mitochondria were isolated from the livers of male Wistar rats using a standard differential centrifugation procedure as described previously [24], using 250 mm sucrose, 10 mm Tris, mm EGTA and mgỈmL)1 BSA (pH 7.7) as the isolation medium Mitochondria were suspended in the medium containing 250 mm sucrose and mm Tris (pH 7.3) Protein was estimated according to Bradford [39] using BSA as the standard Measurement of respiration and membrane potential Prior to each measurement, mitochondria (1 mgỈmL)1 mitochondrial protein) were incubated for with or without lm CdCl2 or lm CuCl2 in the assay medium containing 110 mm KCl, 20 mm Tris, mm KH2PO4, 50 mm creatine, an excess of creatine kinase and mm MgCl2, pH 7.2 Mitochondrial respiration rate and Dw were measured simultaneously at 37 °C in a closed, stirred and thermostated glass vessel equipped with a Clark-type oxygen electrode and TPP+-sensitive electrode, as described previously [40] We used mm succinate (+ lm rotenone) as the respiratory substrate ATP (1 mm) was added to initiate state respiration Data were processed using the chart program supplied with MacLab (AD Instruments, Chalgrove, UK) Determination of CoQ reduction level CoQ reduction level was determined in mitochondria (1 mgỈmL)1 mitochondrial protein) incubated under the conditions used in Dw measurements in a thermostated (37 °C) vessel equipped with platinum and oxygen electrodes, by polarographically measuring the redox state of exogenous CoQ1 (2 lm) [41] To calibrate the platinum electrode traces, Metal-induced impairment of mitochondrial function samples were taken from incubations of mitochondria in standard assay medium without further additions (state 1) and mitochondria were incubated with substrate (5 mm succinate + lm rotenone) One milliliter of sample was quenched with mL of 0.2 m HClO4 in methanol (0 °C) CoQ was extracted with mL of petroleum ether (40–60 °C) and determined by HPLC, as described previously [42] Data were processed using the chart and powerchrom programs supplied with MacLab Determination of SDH activity SDH activity was determined spectrophotometrically at 600 nm from the rate of reduction of DCPIP in the presence of CoQ1, as described in Ragan et al [43] Briefly, intact mitochondria (1 mgỈmL)1 mitochondrial protein) were incubated for with lm CdCl2 or lm CuCl2 under the experimental conditions used in the respiration and Dw measurements Medium was then collected and mitochondrial membranes were ruptured by four freeze–thaw cycles Measurement of SDH activity was carried out in the presence of mgỈmL)1 CoQ1 and 100 lm DCPIP SDH activity was calculated using a molar extinction coefficient of 21 mm)1Ỉcm)1 The dependence of SDH activity on concentrations exceeding lm Cd2+ or Cu2+ ions was determined using a slightly modified protocol: varying amounts of CdCl2 or CuCl2 were added directly to four-times freeze–thawed mitochondrial suspension (i.e without preincubation with intact mitochondria) in an assay medium containing 110 mm KCl, 20 mm Tris, mm KH2PO4, 2.24 mm MgCl2, pH 7.2, and SDH activity was determined as described above Measurement of H2O2 production Oxidation of DCF was used as an indicator of H2O2 production in the mitochondrial matrix Isolated mitochondria were incubated with lm 2¢,7¢-dichlorofluorescin diacetate for 30 Next, mitochondria were resuspended in medium containing 250 mm sucrose, mm Tris (pH 7.3), centrifuged at 7300 g for 10 at °C, and the supernatant discarded To assess H2O2 production, mitochondria (1 mgỈmL)1 mitochondrial protein) were incubated at 37 °C in assay medium (as in SDH activity determination) supplemented with mm succinate and different concentrations of CdCl2 and CuCl2 (0, and 10 lm) The oxidation of DCF was measured spectrofluorimetrically (kex = 485 nm, kem = 535 nm) for using GENios Pro reader (Tecan, Mannedorf, Swită zerland) and the DCF oxidation rate was expressed as relative fluorescence unitsỈ min)1Ỉmg protein)1 To correct for changes in mitochondria-derived background fluorescence, the same measurement was carried out with mitochondria, which were not loaded with 2¢,7¢-dichlorofluorescin diacetate and the rate obtained was subtracted from the DCF oxidation rate FEBS Journal 276 (2009) 3656–3668 ª 2009 The Authors Journal compilation ª 2009 FEBS 3665 Metal-induced impairment of mitochondrial function J Ciapaite et al Assessment of lipid peroxidation Malondialdehyde content was determined from the formation of TBARS using a TBA assay [44] Isolated mitochondria (5 mgỈmL)1 mitochondrial protein) were incubated in a thermostated (37 °C) vessel in mL of assay medium (as in the SDH activity determination) supplemented with mm succinate and different amounts of CdCl2 and CuCl2 (0, and 10 nmolỈmg protein)1) After of incubation, medium with mitochondria was collected, centrifuged at 10 000 g for at °C and resuspended in mL of cold fresh incubation medium The washing step was repeated twice to remove any sucrose present in the mitochondrial suspension because sucrose interferes with the TBA assay [44] Then, mL of TBA (0.5% in 20% of trichloroacetic acid) was added and the mixture was incubated in a boiling water bath for 30 Precipitated protein was removed by centrifugation at 10 000 g for 10 at °C The absorbance of the sample was measured at 532 and 600 nm (to correct for turbidity) and the amount of TBARS formed (expressed as nmol TBARSỈmg protein)1) was calculated using a molar extinction coefficient of 1.56 · 105 m)1Ỉcm)1 Modular kinetic analysis In the first application of the analysis, the mitochondrial oxidative phosphorylation system was conceptually divided into three functional modules based on the production and consumption of Dw: (a) respiratory chain module, (b) phosphorylation module, and (c) proton leak module [21] In this study, we measured mitochondrial Dw instead of proton-motive force (Dp) because we have previously shown that measurement of Dw (one component of Dp) instead of Dp does not introduce significant error This is because the proton concentration gradient (DpH) (the second component of Dp) is small and does not change under our experimental conditions [25] In the second application of the analysis, respiratory chain enzymes were grouped into two functional modules with the fraction of CoQ (XCoQ) as the connecting intermediate: (a) a CoQ-reducing module, and (b) a CoQ-oxidizing module [23] The kinetics of the modules were determined by titration with specific inhibitors and simultaneous measurement of levels of connecting intermediates (Dw or XCoQ) and oxygen consumption rate as a measure of flux, as described previously [21,23] To determine the effects of Cd2+ and Cu2+ ions on the module kinetics, the procedure was repeated in the presence of these substances Metabolic control analysis The control of a pathway flux by a specific enzyme (or group of enzymes, i.e module) can be assessed quantitatively using MCA [17–19] The flux control coefficient (C) 3666 quantifies the importance of each enzyme (module) in the pathway in controlling the flux through that pathway at a steady state It represents the fractional change in the system property (flux, J) in response to an infinitesimal change in the activity of the enzyme (or module) of the system [17–19] In this study, the flux control coefficients of the oxidative phosphorylation modules over respiration (JR) and phosphorylation (JP) flux were calculated from the 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Three -modular kinetic analysis of effects of Cd2+ and Cu2+ ions on oxidative phosphorylation Bimodulular kinetic analysis of the effects of Cd2+ and Cu2+ ions on oxidative phosphorylation To determine... Effect of Cd2+ and Cu2+ ions on the kinetics of the CoQ-reducing and CoQoxidizing modules Effect of Cd2+ on the kinetics of the CoQ-oxidizing module (A) and CoQ-reducing module (B) Effect of Cu2+. .. which oxidative phosphorylation components were affected by Cd2+ and Cu2+ ions in liver mitochondria oxidizing succinate, we first used threemodular kinetic analysis with Dw as the connecting intermediate