Influence of cadmium on oxidative stress and NADH oscillations in mussel mitochondria

Comparative Biochemistry and Physiology, Part C 216 (2019) 60–66

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Influence of cadmium on oxidative stress and NADH oscillations in mussel mitochondria H. Hanana, C. Kleinert, C. André, F. Gagné

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Aquatic Contaminants Research Division, Environment and Climate Change Canada, 105 McGill, Montreal, Québec H2Y 2E7, Canada

A R T I C LE I N FO

A B S T R A C T

Keywords: NADH oscillations Mitochondria Oxidative stress Metallothionein Cadmium

Biological organisms evolved to take advantage of recurring environmental factors which enabled them to assimilate and process metabolic energy for survival. Mitochondria display non-linear oscillations in NADH levels (i.e. wave behavior) that result from the balance between NADH production (aerobic glycolysis) and oxidation for ATP synthesis. The purpose of this study was to examine the effects of cadmium (Cd) on mitochondrial NADH oscillations in quagga mussels Dreissena bugensis exposed to 50 and 100 μg/L CdCl2 for 7 days at 15 °C. Metallothionein (MT) levels, thioredoxin reductase (TrxR) activity and NADH oxidation rate were also determined, as were oscillations in NADH and the formation of dissipative structures (turbidity), in isolated mitochondria suspensions. The results show that exposure to Cd readily induced MT levels at both concentrations tested and that TrxR and NADH oxidase activity was induced at 100 μg/L Cd only. In control mussels, NADH levels oscillated in mitochondria suspensions with a natural period of 2 to 2.5 min for up to 40 min. Exposure to Cd increased the complexity of the frequency profile of NADH oscillations and reduced the amplitudes of the natural signal with a period of 2 to 2.5 min. The formation of dissipative structures decreased in response to a Cd concentration of 100 μg/L but increased at a level of 50 μg/L. The amplitudes at the natural frequency were significantly correlated with NADH oxidase activity (r = −0.91) and with the formation of dissipative structures (r = −0.59). We conclude that Cd could alter the natural frequency in oscillations of NADH in mitochondria, thereby contributing to an increase in NADH oxidation rate and disruption of the spatial organization of mitochondria in suspension. In conclusion, changes in the wave behavior of NADH in mitochondria are proposed as a novel biomarker of toxicity in aquatic organisms.

1. Introduction Biological organisms learned to adapt to recurring environmental changes by adjusting their feeding regime to assimilate and allocate energy for their survival, growth and reproduction. Rapid adaptation is achieved through the development of internal control mechanisms for coping with unstable, variable environments. For example, dynamic control mechanisms may manifest as oscillations, such as cytosolic calcium oscillations (Woods et al., 1987), electrical pacemakers in nerve or cardiac cells (Brown and Guyenet, 1984) and glycolysis activity (Pye and Chance, 1966; Gooch and Packer, 1974). Another example is circadian rhythms (e.g., melatonin), which allow living organisms to adapt to changes caused by Earth's rotation. This type of physiological control offers the advantage of rapid adaptation to environmental changes. To date, few studies have examined the influence of environmental contaminants and climate change on these processes. In particular, the way toxic chemicals alter the non-linear wave patterns

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of biochemical processes is not well understood and has become the subject of an emerging field called chronoecotoxicology or wave ecotoxicology. The effect of contaminants on the wave-like behavior characterizing molecular changes is gaining more and more attention; the strong interest in this avenue of research relates to the fact that many biological processes are cyclical, especially at the sub-cellular and molecular levels (Gagné, 2018). Since the Great Oxidation event some 2.5 billion years ago, many organisms have learned to use molecular oxygen as a means of extracting energy (ATP) from organic matter. A delicate balance is necessary between oxidation of carbohydrates and lipids for the production of ATP during respiration and the concurrent production of highly-reactive oxygen species (ROS), which can damage the cell's environment (Cortassa et al., 2004). Most life forms (prokaryotes and eukaryotes) have thus developed means to synchronize energy production with the handling of ROS. For example, circadian rhythms are found in every living organism and underlie many physiological processes such as redox homeostasis, signal transduction and

Corresponding author. E-mail address: [email protected] (F. Gagné).

https://doi.org/10.1016/j.cbpc.2018.11.005 Received 12 October 2018; Received in revised form 31 October 2018; Accepted 5 November 2018 Available online 07 November 2018 1532-0456/ Crown Copyright © 2018 Published by Elsevier Inc. All rights reserved.

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spectra, suggesting loss of synchronization of the cyclic redox chemical reactions (Gagné et al., 2018). The purpose of the present study was therefore to examine changes in NADH oscillations in mitochondria suspensions from freshwater mussel Dreissena bugensis exposed to cadmium (Cd). The rate of NADH oxidation, oxidative stress and detoxification mechanisms (metallothionein induction) were also measured in order to determine their influence in relation to mitochondrial NADH oscillations in Cd-exposed animals.

xenobiotic metabolism (Levi and Schibler, 2007). However, little is known about whether chemicals can disrupt these cycles and initiate toxicity in cells. Mitochondria are responsible for cellular respiration and energy production, and thus play a pivotal role in energy metabolism. Located at the convergence of most catabolic and anabolic pathways, mitochondria are central to aerobic life processes in organisms. During the formation of the transmembrane proton gradient potential, especially at complex I of the electron transport chain, which drives the production of ATP, ROS are produced (Poynton and Hampton, 2014). Mitochondria produce most of the ROS in cells, which can lead to increased oxidative damage (Balaban et al., 2005). The production of high energy electrons during cellular respirations makes mitochondria highly susceptible to various environmental stressors such as pollution, hypoxia, pH shifts and abrupt changes in temperatures (Sokolova, 2018). Indeed, cellular respiration involves significant oxygen intake, which represents a constant threat to the redox status in cells, and xenobiotics are known to exacerbate this process. Transitions in dissolved oxygen levels can also influence the production of ROS in cells which could lead to oxidative stress (Giannetto et al., 2017). Exposure to air leads to increased expression of superoxide dismutases, including the mitochondrial form (Mn-SOD), catalase and glutathione S-transferase and returned to control levels following re-oxygenation in water. This suggests that air exposure increased antioxidant status in mussels and prevents ROS damage during re-oxygenation. Oscillations in redox intermediates in mitochondria represent an early adaptation to oxygen, which involves coupling between ROS removal and glycolysis to maintain mitochondrial membrane potential, mediated by the ADP/ATP antiporter and the mitochondrial F0F1-ATPase (Olsen et al., 2009). Mitochondria suspensions exposed to a pulse of strontium (a calcium analog efficiently transported across mitochondrial membranes) can trigger sustained oscillations of divalent ions (calcium, magnesium) and NADH (Gylkhandanyan et al., 1976; Aon et al., 2008a) with a period of 2 to 4 min. Oscillations can also be initiated without strontium by adding pyruvate, succinate and ADP (MacDonald et al., 2003). Other intermediates also oscillate in mitochondria such as H+, K+, Ca2+, as do intermediates (citrate) of the citric acid cycle (CAC). These oscillations result from the dynamic control between NADH formation during the CAC and NADH oxidation for ATP synthesis as well as the control of ROS production at complex I of the electron chain transport system (Cortassa et al., 2004). In situations where a loss of balance occurs between NADH production and oxidation in mitochondria, a dampening of the sinusoidal oscillations in NADH can be observed. ROS handling in mitochondria is mainly ensured by peroxiredoxin-thioredoxin proteins in addition to Mn-superoxide dismutase (Poynton and Hampton, 2014). Metallothioneins (MT) are involved in the sequestration of heavy metals and also ROS which could play an important role in mitochondria as well (Gagné et al., 2008; Viarengo et al., 1989). Peroxiredoxins are an important group of proteins with peroxidase activity which are involved in the elimination of H2O2. Thioredoxin reductase activity is required to maintain pyroxiredoxins in the active (reduced) state. Loss of mitochondria synchronization could also lead to decreased amplitudes of NADH waves. Mitochondria have been shown to be organized spatially, forming dissipative structures which are easily measured at 540 nm (turbidity) and associated with synchronization phenomena (i.e., mitochondria are able to oscillate at the same frequency and in phase with one another) (Kurz et al., 2010). The formation of dissipative structures is a common feature where oscillations leading to the formation of visible, spatially organized structures involving calcium waves (Mair and Muller, 1996) or a non-organic analog of the citric acid cycle called the Belousov–Zhabotinsky reaction (Gao et al., 2006). The appearance of these spatially organized structures is a fascinating property whereby mitochondria oscillate in phase and resonate to achieve synchronization. The interaction of chemicals was examined in chemical-based redox oscillators and produced a more complex pattern composed of low amplitude changes in the frequency

2. Materials and methods 2.1. Mussel collection and exposure to Cd Adult quagga mussels (Dreissena bugensis) were collected in August 2017 at a reference site in the St. Lawrence River upstream of the city of Montréal, Quebec, Canada. Clumps of mussels (2–3 cm long) were removed and transferred to the laboratory. They were kept at 4 °C in the dark and in saturated humidity during transport. The quagga mussels were acclimated for 4 to 6 weeks in 50-L aquaria filled with dechlorinated, UV-treated tap water (City of Montreal) at 15 °C on a 16-h light/8-h dark cycle under constant aeration. The mussels were fed 3 times per week with concentrates of phytoplankton (Phytoplex, Kent Marine, WI) and Pseudokirchneriella subcapitata algal preparations (2 L of 100 million cells/mL). The mussels were exposed to 50 and 100 μg/L CdCl2 in 4-L containers lined with polyethylene bags. The Cd concentrations were selected to produce a significant effect on detoxication mechanisms (metallothioneins induction) and oxidative stress in mussels (i.e., a positive control toxicant) based on a previous study (Ivankovic et al., 2010). Each container held N = 28 mussels, and 3 containers were used for each treatment (i.e., 3 control tanks and 3 tanks for each Cd concentration). The mussels were exposed to these conditions for 7 days at 15 °C under constant aeration. The control group consisted of mussels exposed to dechlorinated and UV-treated tap water only. At the end of the exposure time, the mussels were collected in the morning (09:00) and their soft tissues were removed, weighed and homogenized at a 1:5 (w/v) ratio in ice-cold 10 mM Hepes-NaOH (pH 7.4) containing 250 mM sucrose, 1 mM EDTA and 1 μg/mL apoprotinin. Homogenization was performed using a plastic Polytron with one 30-second burst at 5000 rpm. The homogenates were centrifuged at 2000 ×g for 10 min at 2 °C; the resulting supernatant was centrifuged at 10,000 ×g for 20 min at 2 °C and the supernatant (S10 fraction) was collected for determination of thioredoxin reductase (TrxR) activity and metallothioneins (MT) using the method described below. The mitochondria pellet was resuspended in 250 mM sucrose containing 10 mM Hepes-NaOH, pH 7.4, 1 mM KH2PO4 and 1 mM NaHCO3 to measure NAD(P)H oxidase and NADH oscillations in mitochondria. The supernatant (S10) and the mitochondrial fraction were stored at −85 °C until analysis. Concentrations of the biomarkers studied were normalized with the total individual protein concentration according to the Bradford method (Bradford, 1976) using standard solutions of serum bovine albumin for calibration. 2.2. Biomarker analyses Thioredoxin reductase activity (TrxR) was assayed in 50 mM potassium phosphate (pH 7.0) containing 0.5 mM EDTA, using a microplate-based spectrophotometric method (Tedesco et al., 2010). In the presence of 0.5 mM NADH, TrxR reduces 5,5′‑dithiobisnitrobenzoic acid (DTNB, 1 mM) to produce 2‑nitro‑5‑thiobenzoate which can be monitored at 412 nm for 30 min in a plate reader (Synergy-4, USA). As DTNB can also react with glutathione reductase and glutathione peroxidase, the assay was performed in the presence/absence of a specific inhibitor of TrxR, that is, aurothiomalate (ATM) at 20 μM, thus allowing specific TrxR activity to be determined. Enzymatic activity was expressed as nmoles of TNB formed min−1 mg−1 of protein. 61

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Fig. 1. Induction of metallothionein and thioredoxin reductase in mussels exposed to Cd. The levels of MT and thioredoxin reductase activity were determined in mussels exposed to Cd. The data are expressed as the mean with standard error. The star symbol * represents significance from controls at p = 0.05.

prepared as described above but placed in clear microplates for the measurement of absorbance at 540 nm, which was taken every 30 s for 40 min as described above without agitation. The data were expressed as the ratio of the maximal increase at 540 nm to the minimum value at t = 0.

Total MT levels were evaluated using the non-radioactive silversaturation assay (Scheuhammer and Cherian, 1986; Gagné et al., 1990). Briefly, the S10 fraction was saturated with 5 μg/mL silver at pH 8.5 (Ag+ in 100 mM glycine buffer) for 5 min, then 2% bovine hemoglobin was added for 10 min to remove loosely bound silver; this was followed by heat denaturing at 100 °C for 2 min and centrifugation at 10,000 ×g to remove Ag-hemoglobin and heat-denatured proteins. The supernatant was subjected again to the hemoglobin addition/heat denaturation and centrifugation steps, and silver remaining in the supernatant was determined by graphite furnace atomic absorption spectrophotometry with Zeeman effect background correction. A ratio of 12 mol of bound Ag to 1 mol of MT was assumed for mussel MT (Kille et al., 1994). Results were expressed as nmoles of MT equivalent mg−1 of protein. The oxidation rate of NADH (NADH oxidase activity) was determined in isolated mitochondria. The mitochondria suspension was diluted to 0.1 mg/mL in 250 mM sucrose, 10 mM tris-HCl, pH 7.5, 0.1 mM MgCl2, and 1 mM KCl. After 5 min, 0.1 mM NADH was added to the suspension, and fluorescence measurements at 360 nm excitation and 460 nm emission were taken every 5 min for 30 min at 30 °C. The assay was performed in 200 μL volume in dark microplates with a fluorescent microplate reader (Synergy-4, USA). The oxidation rate was reported as a decrease in NADH fluorescence min−1 mg−1 of protein. Oscillations between reduced NAD(P)H and oxidized NAD(P)+ in mitochondria were also measured in dark 96-well microplates. Mitochondria were diluted to 0.1 mg/mL total protein in the reaction media composed of 200 mM Mannitol, 50 mM sucrose, 5 mM KH2PO4, 5 mM NaHCO3, 2 mM MgCl2, 1 mM K-ADP, 2 mM pyruvate and 5 mM Hepes-NaOH (pH 7.4). Microplate fluorescence readings were taken every 30 s for 40 min in sweep mode (excitation using a quick flash burst of 10 ms and emission readings with no delay) using the Synergy4 microplate reader at 360 nm and 460 nm for excitation and emission wavelengths, respectively. Instrument calibration was achieved with blank solution (composed of the reaction media only) and standard additions of NADH at 50 μM in the reaction media. The formation of dissipative structures was determined by measuring the turbidity of the suspension at 540 nm (Holmuhamedov, 1986). Mitochondria were

2.3. Data analysis The experiment was repeated 3 times using 28 mussels for each Cd exposure treatment and controls. The data were subjected to normality and variance homogeneity testing using the Shapiro-Wilks and BrownForsyth tests, respectively. The data were then subjected to analysis of variance with the Cd exposure concentration (0, 50 and 100 μg/L) as the main effect (N = 10 mussels per treatment group, 9 degrees of freedom or df). Critical differences between groups were confirmed by the Fishers Least Square Difference test. Correlations between biomarkers were determined using the Pearson moment correlation test. A Fourier transformation analysis was performed on mitochondrial NADH oscillations to obtain the periodogram values, which represent the variance at a given frequency. Briefly, Fourier transformation consists in finding the sine and cosine functions of the data: g(w) = a0 + ∑(Asin (wt) + Bcos(wt)), where w is the frequency and t is time (discrete Fourier transformation). The coefficients A and B are related to the amplitude of changes and are used to obtain the periodogram value (which is the square sum of A and B and represents the variance of the amplitude at each frequency signal). All tests were performed using Statistica (version 13, TIBCO software Inc., USA) and the significance was set to p = 0.05 in all cases. 3. Results Quagga mussels were exposed to Cd (50 and 100 μg/L) for 7 days at 15 °C and analyzed for the activation of metal detoxification and oxidative stress mechanisms (Fig. 1). MT levels were significantly induced at both Cd concentrations, reaching levels > 4 times those found in the controls (ANOVA p < 0.001, df = 9). TrxR activity was significantly induced at the highest Cd concentration, reaching levels nearly 1.5 62

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In mussels exposed to 50 μg/L Cd, the oscillations in NADH levels were somewhat similar to those observed in controls but the NADH peaks appeared smaller and the distance between peaks higher relative to the controls (Fig. 4A). In mussels exposed to 100 μg/L Cd, this effect was greatly enhanced, i.e., decreased amplitudes and lower frequency. This was confirmed by periodogram analysis (Fig. 4B). The periodogram value at the normal frequency of 0.2 to 0.25 shifted to lower frequencies and showed decreased values at 50 μg/L Cd, followed by the absence of any signals at this frequency range for mussels exposed to 100 μg/L Cd. At the other frequency 0.13, the frequency was shifted to a lower frequency with no changes in periodogram values in mussels exposed to 50 μg/L Cd. The signal at frequency 0.13 was decreased in mussels exposed to 100 μg/L Cd, with an apparent shift at a higher frequency of 0.175. During oscillations, mitochondria were observed to form dissipative structures which increase the density at 540 nm (Holmuhamedov, 1986). This increase in “turbidity” is associated with the organization of mitochondria networks in suspension, which points to mitochondria crosstalk capacity whereby mitochondria oscillations are synchronized within the cell environment (Fig. 5). The maximal change in the absorbance at 540 nm was significantly increased in mussels exposed to 50 μg/L Cd, followed by a decrease at 540 nm in mussels exposed to 100 μg/L Cd which suggests lost of communication between mitochondria. Correlation analysis was performed (Table 1) to determine the relationships between mitochondria oscillations, NADH oxidation rate and oxidative stress. The analysis revealed that the NADH oxidation rate was negatively related with absorbance at 540 nm (r = −0.59; p < 0.05) and with periodogram values at frequency 0.23 (r = −0.91; p = 0.001), which suggests that a decrease in in NADH oscillations is associated with an increase in the oxidation rate of NADH levels in mitochondria suspensions. The periodogram values were correlated with MT levels (r = −0.68; p < 0.05) and marginally correlated with TrxR activity (r = −0.65; p = 0.06). Canonical analysis showed that the oscillatory behavior (periodogram and absorbance at 540 nm) was highly correlated with MT, TrxR and NADH oxidation rate (r = 0.86 [p < 0.001]), which suggests that oxidative stress was related to changes in oscillatory behavior in mitochondria in Cd-exposed mussels.

Fig. 2. Change in NADH oxidase activity in mitochondria from mussels exposed to Cd. The oxidation rate of NADH was followed in mitochondria suspensions from mussels exposed to Cd. The data are expressed as the mean with standard error. The star symbol * represents significance from controls at p = 0.05.

times those found in controls. This suggests that oxidation of peroxidoxins in mitochondria occurs at a Cd concentration of 100 μg/L. The oxidation rate of NADH was also measured in isolated mitochondria (Fig. 2). NADH oxidase activity was significantly induced at the highest Cd concentration (100 μg/L), reaching 1.5-fold induction relative to controls. NADH oxidase activity was significantly correlated with TrxR, which also depends on NADH for its activity (r = 0.65; p < 0.05). A closer examination using covariance analysis of NADH oxidase activity revealed that the exposure Cd concentration was a more significant factor (p < 0.001) than TrxR activity (p = 0.4) which suggests that TrxR activity was not a major contributor to the NADH oxidase activity in mitochondria. In controls, NADH levels oscillate over time in the presence of cofactors such as ADP and pyruvate in mitochondria suspensions (Fig. 3A). A characteristic oscillation occurs at a frequency of 0.2 to 0.25 which corresponds to periods of 2 to 2.5 min (Fig. 3B), a pattern also reported in mammalian and yeast mitochondria (Gooch and Packer, 1974). Another oscillation signal was also observed, at frequency 0.13 corresponding to a period of 4 min. The oscillatory behavior of NADH levels was also examined in mussels exposed to Cd. 2930

A

4. Discussion Exposure of mussels to Cd leads to induction of MT levels and TrxR activity, which points to the activation of a mechanism of detoxification of divalent metals and protection against oxidative stress. The induction of MT by Cd is a well-known response involved in protection against

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2830 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85

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0

5

Periodogram Value

NAD(P)H levels (Relative Fluorescence units)

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0 0,00

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Frequency (1/30 sec observation)

Observation (30 sec)

Fig. 3. Oscillatory change in NAD(P)H levels in mitochondria suspension. Mitochondria were isolated from the soft tissues of control mussels and incubated with pyruvate and ADP to initiate NADH oscillations. Time-dependent changes in NADH levels (A) and frequency analysis using Fourier transformation are shown (B). 63

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Fig. 4. Effect of Cd exposure on the oscillatory changes in NAD(P)H fluorescence in mussels. Mitochondria were isolated from the soft tissues of Cd-exposed mussels and incubated with pyruvate and ADP to initiate NADH oscillations. Time-dependent changes in NADH levels (A) and frequency analysis using Fourier transformation are shown (B) with increasing Cd concentrations.

which are continuously produced in mitochondria during respiration and energy production (ATP) (Gagné et al., 2007, 2008). It was shown that pre-treating mussels with Cd could prevent oxidative stress when mussels were challenged with iron or when isolated digestive gland cells were treated with H2O2 (Viarengo et al., 1999). Mitochondria contain high levels of peroxiredoxins, which contain cysteinyl residues (SH) responsible for the breakdown of H2O2 → 2H2O + O2. During this process, reduced cysteines in peroxiredoxins are oxidized to sulfenic acid (RSOH), which is reduced by reduced thioredoxins. TrxR activity, which is involved in the maintenance of reduced thioredoxins, was induced at the highest Cd concentration (100 μg/L). The induction of TrxR activity at the highest Cd concentration suggests that MT was not able to prevent oxidative stress in mitochondria on its own. However, TrxR activity was significantly correlated with the NADH oxidation rate in mitochondria (r = 0.65; p < 0.05), which is indicative of electron chain activity producing ROS during the oxidation of NADH. Oscillations in NADH levels in mitochondria result from the equilibrium between the oxidation of NADH during electron transport activity which supports the transmembrane H+ gradient for ATP synthesis and the production of NADH during the CAC. Complex I of the electron transport chain is responsible for most of the NADH oxidase activity and the production of ROS (Poynton and Hampton, 2014). In the presence of cofactors such as pyruvate and ADP, mitochondria produce NADH during aerobic glycolysis, which provides electrons for the electron transport chain starting at complex I. The transfer of high energy electrons at this step also produces ROS, which are neutralized by the peroxiredoxin/thioredoxin/TrxR system. Another NADH oxidase enzyme is involved in the maintenance of complex I which could account for NADH oxidation in mitochondria (Norberg et al., 2008). This

Fig. 5. Change in turbidity in mitochondria suspension from mussels exposed to Cd. The appearance of dissipative structures during mitochondria oscillations was measured at 540 nm. The maximum increase at 540 nm is reported during the oscillations. The star symbol * represents significance from controls at p = 0.05.

toxic heavy metals (Kille et al., 1994). MT is also induced in molluscs through exposure to Cd; it participates in detoxification by binding the metal (Roesijadi et al., 1989; Ivankovic et al., 2010). In mussels, MT also plays a role in the sequestration of reactive oxygen species (ROS) Table 1 Correlation analysis. Variable

NAD(P)H oxidation rate

Aggregation indexa

Thioredoxin reductase

MT

Frequency 0.23 (periodogram)

NAD(P)H oxidation rate

1

r = −0.59 p < 0.04

r = 0.65 p < 0.05 r = −0.36 p > 0.1 1

r = 0.49 p = 0.1 r = 0.20 p < 0.1 r = 0.23 p > 0.1 1

r = −0.91 p = 0.001 r = 0.49 p > 0.1 −0.65 0.1 < p < 0.5 r = −0.68 p < 0.05

Aggregation index

1

Thioredoxin reductase MT

a

Aggregation index: maximal absorbance at 540 nm during oscillations. 64

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activity in quagga mussels exposed to Cd levels of 50 and 100 μg/L for 7 days. In mitochondria, the oxidation rate of NADH was significantly increased at the highest Cd concentration. The effect of Cd on the fundamental property of mitochondrial NADH oscillations revealed that the normal frequency of NADH oscillations corresponding to a period of 2 to 2.5 min could be altered at Cd concentration before the impacts on NADH oxidase activity are observed. The data suggest that an alteration in the frequency profile of NADH oscillations appears to manifest at Cd concentrations where the detoxification mechanism comes into play (MT), as evidenced by the significant correlation between the amplitude with a period of 2 to 2.5 min and MT levels. The effects of chemicals on the fundamental wave-like behavior of biochemical processes should be examined more closely since oscillators are involved in the integration of energy production/respiration and ROS control in cells. Future research are under way to determine whether wild organisms exposed long-term to contaminants are affected in respect to the oscillatory behavior in biochemical processes involved in energy production.

enzyme serves also as an apoptosis inducing factor which depends on the release of free Ca2+ which also regulates key enzymes in the CAC (Bertram et al., 2009). It is noteworthy that NADH oxidase activity was negatively correlated with the aggregation index (turbidity determined at 540 nm) and periodogram value at the normal of frequency of 0.2 to 0.25 (NADH changes with a period of 2 to 2.5 min). This suggests that high NADH oxidase activity and MT levels could dampen NADH oscillations in mitochondria. In other words, when NADH oxidase activity is increased, the balance between NADH production and elimination and ROS handling is disrupted. Moreover, the aggregation of mitochondria forming dissipative structures in suspensions was also limited during oxidative stress as determined by high levels of MT and NADH oxidase activity when the intensity of oscillations at a frequency of 0.2 to 0.25 was dampened. At low Cd concentration (50 μg/L), the formation of dissipative structure was enhanced. A possible explanation for this is that enhanced mitochondria ROS increase synchronization between mitochondria in suspension. This suggests that the toxic action of Cd results in an increase in oxidative stress, by the higher TrxR activity and NADH oxidase activity. We found no evidence of the effect of Cd during the CAC during the production of NADH. However, ROS production could inhibit the activity of aconitase, an enzyme in the CAC involved in the isomerization of citrate to isocitrate in oysters exposed to Cd at high temperatures (Cherkasov et al., 2007). The inhibition appeared to be more closely associated with the production of ROS rather than due to the direct action of Cd on aconitase. The formation of complex oscillatory redox activity forms the basis of normal mitochondrial function and departure to pathophysiological state (Kembro et al., 2014). By modulating the levels of antioxidant enzymes and ROS levels, cells oscillate between energy production and ROS removal. In conditions with high ROS levels, more complex waveforms in NADH levels were observed. This was corroborated by the formation of NADH changes at other frequencies in mussels exposed to Cd. As suggested by the strong canonical correlation results for oscillatory properties of mitochondria (NADH oscillations and dissipative structures) and redox markers (TrxR and MT), the control of ROS levels through oscillatory activity in mitochondria represents an adaptive strategy for balancing energy production and oxidative stress. ROS also oscillates with NADH levels in mitochondria in normal conditions. When an external agent (e.g., xenobiotic) disrupts this process and stimulates ROS production, the natural cycles of NADH and ROS production are disrupted and toxicity is initiated. ROS levels in mitochondria also exhibit wave-like behavior during the production of ATP (complex I of electron chain transport) (Aon et al., 2008b), which means that chemicals contributing to ROS production could disrupt the regulation of ROS levels and hence reduce its synchronicity with NADH oscillations. Mitochondrial oscillations are ROS-dependent which results from the interplay between ROS production, transport and scavenging, including CAC, oxidative phosphorylation and Ca2+ handling (Zhou et al., 2010). Among the major ROS, superoxide diffusion is locally handled by anion transporters at the inner membrane and Mn-superoxide dismutase in the matrix of mitochondria. The use of 4‑chlorodiazepam to decrease the activity of this anion transport system in mitochondria dampened the oscillations in ROS and NAD(P)H in rat cardiomyocytes and yeast cells (Aon et al., 2008a). The oscillatory behavior in NADH and redox homeostasis represents a fundamental way cells integrate their cellular functions while providing inertia in response to environmental external agents. Cd also has the ability to decrease total SOD activity (Cu, Zn- and Mn-superoxide dismutase) in carp erythrocytes exposed to 20 mg·L−1 Cd after 12 h (Zikic et al., 1997). Cd could lead to the formation of ROS through a Fenton-like reaction resulting in the production of superoxide anion, H2O2 and hydroxyl radicals (Stohs and Bagchi, 1995). Fenton-like reactions may be commonly associated with membranous structures such as mitochondria, microsomes and peroxisomes since they have high levels of hemoproteins (iron acts as a catalysis). In conclusion, exposure to Cd leads to induction of MT and TrxR

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