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Aquatic Toxicology 158 (2015) 75–87

Contents lists available at ScienceDirect

Aquatic Toxicology

j ourna l ho me pa ge: www.elsev ier .com/ locate /aquatox

odulation of cadmium-induced mitochondrial dysfunction andolume changes by temperature in rainbow trout (Oncorhynchusykiss)

ohn O. Onukwufora, Fred Kibengeb, Don Stevensa, Collins Kamundea,∗

Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE, Canada C1A 4P3Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE, Canada C1A 4P3

r t i c l e i n f o

rticle history:eceived 22 September 2014eceived in revised form 3 November 2014ccepted 5 November 2014vailable online 13 November 2014

eywords:admiumemperaturenteractions

itochondrial bioenergeticsitochondrial volume

ainbow trout

a b s t r a c t

We investigated how temperature modulates cadmium (Cd)-induced mitochondrial bioenergetic dis-turbances, metal accumulation and volume changes in rainbow trout (Oncorhynchus mykiss). In the firstset of experiments, rainbow trout liver mitochondrial function and Cd content were measured in thepresence of complex I substrates, malate and glutamate, following exposure to Cd (0–100 �M) at three(5, 13 and 25 ◦C) temperatures. The second set of experiments assessed the effect of temperature on Cd-induced mitochondrial volume changes, including the underlying mechanisms, at 15 and 25 ◦C. Althoughtemperature stimulated both state 3 and 4 rates of respiration, the coupling efficiency was reduced attemperature extremes due to greater inhibition of state 3 at low temperature and greater stimulation ofstate 4 at the high temperature. Cadmium exposure reduced the stimulatory effect of temperature onstate 3 respiration but increased that on state 4, consequently exacerbating mitochondrial uncoupling.The interaction of Cd and temperature yielded different responses on thermal sensitivity of state 3 and 4respiration; the Q10 values for state 3 respiration increased at low temperature (5–13 ◦C) while those forstate 4 increased at high temperature (13–25 ◦C). Importantly, the mitochondria accumulated more Cdat high temperature suggesting that the observed greater impairment of oxidative phosphorylation withtemperature was due, at least in part, to a higher metal burden. Cadmium-induced mitochondrial vol-ume changes were characterized by an early phase of contraction followed by swelling, with temperature

changing the kinetics and intensifying the effects. Lastly, using specific modulators of mitochondrial ionchannels, we demonstrated that the mitochondrial volume changes were associated with Cd uptake viathe mitochondrial calcium uniporter (MCU) without significant contribution of the permeability transi-tion pore and/or potassium channels. Overall, it appears that high temperature exacerbates Cd-inducedmitochondrial dysfunction and volume changes in part by increasing metal uptake through the MCU.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

In a natural environment aquatic ectotherms, such as fish,re particularly vulnerable to changes in temperature becauseheir body temperatures are close to that of the environmentStevens and Fry, 1974). Indeed, major physiological and bio-

hemical processes in fish, including swimming, metabolic rate,rowth and reproduction are highly affected by temperature fluc-uations. To cope with environmental temperature change, aquatic

∗ Corresponding author at: Department of Biomedical Sciences, Atlantic Vet-rinary College, University of Prince Edward Island, 550 University Avenue,harlottetown, PE, Canada C1A 4P3. Tel.: +1 902 566 0944; fax: +1 902 566 0832.

E-mail address: ckamunde@upei.ca (C. Kamunde).

ttp://dx.doi.org/10.1016/j.aquatox.2014.11.005166-445X/© 2014 Elsevier B.V. All rights reserved.

organisms have evolved a wide array of mechanisms. In fish manyof these mechanisms entail modulation of energy metabolism andinclude changes in mitochondrial membrane properties, densityand enzyme activity (Guderley and St-Pierre, 2002; Kraffe et al.,2007; Lockwood and Somero, 2012; Oellermann et al., 2012).Within a zone of tolerance, these changes allow organisms tocope with the challenges associated with extreme temperatures.Because the mitochondria perform several other important func-tions such as cell signalling, redox regulation, Ca homeostasis andcontrol of apoptosis, temperature-induced mitochondrial dysfunc-tion typically leads to loss of the cell function with cell death as the

terminal sequel.

Environmental temperature stress is commonly encounteredtogether with chemical pollutants including metals such as Cd. Cad-mium is an important trace metal contaminant in aquatic systems

7 tic To

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6 J.O. Onukwufor et al. / Aqua

ue to its environmental persistence and high toxicity to aquaticrganisms (Byczkowski and Sorenson, 1984; Hattink et al., 2005).hen present at elevated levels in aquatic systems, Cd is readily

aken up and accumulated in tissues of resident organisms resultingn toxicity (Kraemer et al., 2005, 2006). Although the toxic effectsf Cd are numerous, the mitochondria are arguably one of the mostmportant target sites of its toxic action. In this regard, severaluthors have demonstrated that many aspects of mitochondrialunction are compromised by Cd in plants (Kesseler and Brand,994a), mammals (Belyaeva and Korotkov, 2003), invertebratesKurochkin et al., 2011) and fish (Adiele et al., 2012; Onukwufort al., 2014). The mechanisms through which Cd alters mitochon-rial function include, formation of complexes with thiol proteinsnd the displacement of iron and/or Cu from their binding sites iney proteins of the respiratory chain (Rikans and Yamano, 2000;orta et al., 2003).

Although it is evident from the foregoing that temperaturend Cd affect cell/organismal function individually, our knowl-dge of their combined effects is limited to few studies thatave to date failed to generate a consistent theme regardingemperature–metals interactions. For example, when Daphniaagna were exposed to Cd at increasing temperatures, the median

nd threshold lethal body burdens decreased suggesting that loweretal accumulation was needed to kill daphnids at higher tempera-

ure (Heugens et al., 2003). In contrast, Lannig et al. (2006) workingith the eastern oyster (Crassostrea virginica) reported that expo-

ure to Cd at 28 ◦C caused significantly higher mortality comparedith exposure at 20 or 24 ◦C, even though both groups had the same

issue Cd burdens. With regard to interactions on energy homeo-tasis, Sokolova (2004) found that oyster mitochondria were moreensitive to Cd at high temperatures and concluded that temper-ture sensitizes mitochondria to Cd. Thus, the existing literatureuggests that aquatic organisms may be vulnerable to metals lev-ls that ordinarily would not be toxic in the absence of thermaltress but the increased sensitivity is not always associated withigher metal loads.

The mechanisms through which thermal stress moderatesetals-induced mitochondrial dysfunction have not been clearly

lucidated but may include changes in mitochondrial volume.itochondrial volume homeostasis is critical for mitochondrial

unction (Kaasik et al., 2007) and many physiological and patho-hysiological conditions are known to impose volume changes onhese organelles (Guerrieri et al., 2002; Fujii et al., 2004). Underormal physiological conditions the mitochondrial matrix volume

s regulated by the inner mitochondrial membrane (IMM) whichcts as the main barrier for molecules moving into and out ofhe organelle. However, the IMM is endowed with ion exchang-rs, uniporters and channels that impart selective permeabilityo specific molecules (Bernardi, 1999; O’Rourke, 2000; Lee andhevenod, 2006). This tight control of IMM permeability enableshe mitochondria to create a high proton gradient that drives theroduction of ATP that is important for cellular function, main-enance and viability. Alteration in IMM permeability leads tonguarded passage of solutes and water, disrupting the func-ion of the mitochondria (Belyaeva et al., 2001; Li et al., 2003;rlov et al., 2013). Cadmium and temperature stress may alter

he permeability of IMM to solutes resulting in matrix volumehanges. Indeed, evidence of Cd-induced mitochondrial volumehanges has been provided, including swelling and contractionn mammals (Lee et al., 2005a), moderate swelling in rainbowrout (Adiele et al., 2012) and contraction in oysters (Sokolova,004). These varied observations suggest that additional studies are

equired to clarify the mechanisms of Cd-induced mitochondrialolume changes. Furthermore, although there is evidence that tem-erature causes mitochondrial volume changes (Richardson andappel, 1962; He and Lemasters, 2003), little is known about the

xicology 158 (2015) 75–87

combined/interactive effects of thermal stress and Cd on mitochon-drial volume.

In the present study the effect of temperature on Cd-inducedmitochondrial dysfunction and volume changes were investigated.First, we sought to unveil the interactive effects of Cd and tem-perature on oxidative phosphorylation (OXPHOS) by testing theprediction that high temperature will increase mitochondrial Cdaccumulation resulting in greater OXPHOS impairment. Second,given the importance of mitochondrial volume homeostasis indetermining their function, the mechanisms of Cd-induced mito-chondrial volume changes and the effect of temperature on thisphenomenon were investigated. Our findings highlight how tem-perature change in the context of the global climate change incombination with metals, in this case Cd, would impact energyhomeostasis in fish.

2. Materials and methods

2.1. Ethics

All experimental procedures were approved by the Universityof Prince Edward Island Animal Care Committee in accordance withthe Canadian Council on Animal Care.

2.2. Experimental animals

Rainbow trout were obtained from Ocean Farms Inc., Brookvale,PE, and maintained at the Atlantic Veterinary College Aquatic Facil-ity in a 250-l tank containing aerated well-water with temperatureof 13 ± 1 ◦C and a pH of 7.7. The fish were fed at 1% of their bodyweight daily with commercial trout chow pellets (Corey Feed Mills,Fredericton, NB) until sampled to isolate liver mitochondria usedin the experiments. Fish weight ranged from 506 to 560 g duringthe experimental period.

2.3. Mitochondrial isolation

Fish were sacrificed by a sudden blow to the head and dissectedto remove the livers for mitochondrial isolation according to themethod of Onukwufor et al. (2014). Briefly, the livers were rinsedwith mitochondrial isolation buffer (MIB: 250 mM sucrose, 10 mMTris–HCl, 10 mM KH2PO4, 0.5 mM EGTA, 1 mg/ml BSA [free fattyacid], 2 �g/ml aprotinin, pH 7.3), blotted dry and weighed. The liv-ers were then diced and homogenized in 1:3 (weight to volume)ratio of liver to MIB in a 10-ml Potter-Elvehjem homogenizer (ColeParmer, Anjou, QC). Three passes of the pestle mounted on a hand-held drill (MAS 2BB, Mastercraft Canada, Toronto, ON) running at200 rpm were optimal for rainbow trout liver mitochondria isola-tion. The homogenate was then centrifuged at 800 × g for 15 minat 4 ◦C. The supernatant was collected, centrifuged at 13,000 × g for10 min at 4 ◦C and the pellet (mitochondria) was washed twice byre-suspending in MIB and centrifuging at 11,000 × g for 10 min at4 ◦C. The pure mitochondrial pellet was finally re-suspended in a1:3 (weight to volume) ratio of mitochondrial respiration buffer[MRB: 10 mM Tris–HCl, 25 mM KH2PO4, 100 mM KCl, 1 mg/ml BSA(fatty acid free), 2 �g/ml aprotinin, pH 7.3]. The protein content ofthe isolated mitochondrial was measured by spectrophotometry(Spectramax Plus 384, Molecular Devices, Sunnyvale, CA) accordingto Bradford (1976).

2.4. Mitochondrial respiration

Mitochondrial respiration was measured using Clark-type oxy-gen electrodes (Qubit Systems, Kingston, ON) in 1.5 ml cuvettesafter a two-point calibration at 0 and 100% oxygen saturation.A traceable digital barometer (Fisher Scientific, Nepean, ON) was

J.O. Onukwufor et al. / Aquatic Toxicology 158 (2015) 75–87 77

Table 1Summary of two-way ANOVA for mitochondrial respiration indices and Cd accumulation.

Main effect

Parameter Cadmium (Cd) Temperature (T) Interaction (Cd × T)

df F P df F P df F P

State 3 5,72 222 <0.0001 2,72 671 <0.0001 10,72 18 <0.0001State 3 Q10 5,48 1.08 0.3847 1,48 558 <0.0001 5,48 5.6 <0.001State 4 5,72 114 <0.0001 2,72 2455 <0.0001 10,72 53 <0.0001State 4 Q10 5,48 47 <0.0001 1,48 2238 <0.0001 5,48 21 <0.0001P/O ratio 3,48 22 <0.0001 2,48 76 <0.0001 6,48 9 <0.0001

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perature” and “treatment” or “Cd dose” were the independent

RCR 5,72 919 <0.0001 2,72

[Cd] 5,81 357 <0.0001 2,81

sed to measure the atmospheric pressure and temperature wasonitored and maintained at 5, 13 or 25 ◦C with the aid of a recir-

ulating water-bath (Haake, Karlsruhe, Germany). Following thealibration, 1.45 ml of MRB and 100 �l of mitochondrial suspen-ion containing 2.2–2.4 mg of protein (22–24 mg of mitochondrialass, wet weight) were loaded into cuvettes and continuously

tirred. Complex I substrates (5 mM malate and 5 mM glutamate)nd 250 �M ADP were added to initiate state 3 respiration whichransitioned to state 4 upon depletion of the ADP. Lastly, 2.5 �g/mlligomycin was added to inhibit ATP synthase activity in order toeasure state 4ol, a metric of mitochondrial proton leak (Brand

t al., 1994; St-Pierre et al., 2000). To measure the combined effectf Cd and temperature, mitochondria isolated from each fish wereested at 5, 13 and 25 ◦C using 0, 5, 10, 20, 50 and 100 �M Cdor a total of n = 5 per temperature. Because the respiration ratesaried greatly across the three temperatures, Cd exposure timesere synchronized by varying the durations of state 4 and state

ol. Thus, readings at all temperatures were taken after 2, 8 and6 min of Cd addition for state 3, 4 and 4ol respiration rates, respec-ively, thereby allowing valid comparison of the data. Note that theuvettes remained well-oxygenated and were never hypoxic after6 min of Cd addition at the three temperatures. LabPro data acqui-ition software (Qubit Systems) was used to record and analyzell the oxygen consumption data. The phosphorylation efficiencyP/O ratio: ADP used/oxygen consumed) and the respiratory controlatio (RCR: ratio of state 3 to state 4 respiration) were calculatedccording to Estabrook (1967) and Chance and Williams (1955),espectively.

The temperature coefficients (Q10 values) for state 3 and 4 res-irations were calculated for the temperature ranges 5–13 and3–25 ◦C using the van’t Hoff equation: Q10 = (R2/R1)[10/(T2−T1)] (e.g.,ontagnes et al., 2003), where R2 and R1 represent mitochondrial

xygen consumption rates at two temperatures, T2 and T1, andhere T2 > T1.

.5. Mitochondrial Cd content analysis

Cadmium accumulation in the mitochondria was measuredy atomic absorption spectrophotometry (AAS: PinAAcle 900T,erkin Elmer, Woodbridge, ON). Briefly, after measuring the res-iration rate, mitochondria samples were removed from theuvettes and centrifuged at 10,000 × g for 5 min at 4 ◦C. Theupernatants were discarded and the pellets were washed with00 �l of MIB to remove non-specifically bound Cd, with pel-

eting at 10,000 × g for 5 min at 4 ◦C. The pellets were thentored at −80 ◦C until analyzed for Cd. For AAS, the pellets wereven-dried to constant weight at 70 ◦C (ISOTEMP, Fisher Scien-

ific), weighed and digested with 500 �l of 30% H2O2 and 70%NO3 (trace metal grade, Fisher) in a 1:15 mixture for 24 ht room temperature. The digests were diluted appropriatelysing 0.2% HNO3 and the Cd concentrations were measured by

657 <0.0001 10,72 24 <0.00017.85 <0.001 10,81 3.6 <0.001

AAS in furnace mode and expressed as �mol Cd/g mitochondrialdry weight (mdw). All Cd analyses were done in the presenceof modifiers (NH4H2PO4 and Mg (NO3)2). Standard referencematerial (SRM: TMDA-70.2) and blanks were analyzed concur-rently with the samples. Cadmium was not detected in blanksand the recovery rate of Cd from the SRM ranged between 95and 106%.

2.6. Mitochondrial volume

Mitochondrial volume changes were measured as described bySappal et al. (2014). Briefly, mitochondria isolated as describedabove were re-suspended as 1 mg/ml protein in swelling buffer(100 mM KCl, 10 mM Tris–HCl, 25 mM KH2PO4, 1 mg/ml BSA, 5 mMglutamate and 5 mM malate adjusted to pH 7.3). Volume changeswere then measured at 15 and 25 ◦C using 0, 5, 50 and 100 �M Cd,with 200 �M Ca (positive control) as inducers. The cations (Cd orCa) doses were added to microplate wells as 20 �l of appropriatestock solutions and brought to assay volume of 200 �l by adding180 �l of the 1 mg/ml mitochondrial suspension equilibrated totest temperature. The changes in absorbance at 540 nm, wherein adecrease indicates swelling and an increase indicates contraction,were then monitored every 10 s for 30 min. An additional studytested the effect of heat shock on Cd-induced mitochondrial volumechanges by loading the 1 mg/ml mitochondrial suspension directly(i.e., without equilibration to assay temperature) from ice (4 ◦C) tomicroplate wells at 15 or 25 ◦C with the absorbance at 540 nm beingmonitored as above.

To unveil the mechanisms of Cd-induced volume changes, theeffects of modulators of the mitochondrial permeability transitionpore (MPTP: cylosporin A (CsA), 1 �M), mitochondrial calcium uni-porter (MCU: ruthenium red, 5 �M) and mitochondrial potassiumchannels (mitoKATP: diazoxide, 100 �M and 5-hydroxydecanoate(5-HD), 400 �M) on Cd- and Ca-induced volume changes wereassessed. Here, the modulators were added to the wells as 10 �l ofstock solutions and pre-incubated with 170 �l of 1 mg/ml of mito-chondrial suspension for 5 min, after which 100 (Cd) or 200 (Ca) �Mwere added as 20 �l of appropriate stock solutions. Absorbancechanges at 540 nm were then monitored every 10 s for 30 min at15 and 25 ◦C.

2.7. Data analysis

The data were tested for normality of distributions (chi-squaretest) and homogeneity of variances (Cochran C) then submit-ted to one or two-way analysis of variance (ANOVA) (Statisticaversion 5.1, Statsoft, Inc., Tulsa, OK). In these analyses, “tem-

variables. Significantly different means were separated usingTukey’s post hoc test at P < 0.05. Linear regression analysis wasperformed using SigmaPlot 10 (Systat Software, San Jose, CA,USA).

78 J.O. Onukwufor et al. / Aquatic Toxicology 158 (2015) 75–87

Fig. 1. Interactive effects of temperature and Cd on (a) state 3 respiration and (b)Q10 of state 3. Mitochondria isolated from each fish were exposed to Cd (0, 5, 10,2 ◦

dT

3

3

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Fig. 2. Interactive effects of temperature and Cd on (a) state 4 respiration, and (b)Q10 of state 4. Mitochondria isolated from each fish were exposed to Cd (0, 5, 10,20, 50 and 100 �M) at 5, 13 and 25 ◦C. Data are means ± SEM (n = 5). Points withdifferent letters are statistically different from each other (two-way ANOVA withTukey’s HSD, P < 0.05).

0, 50 and 100 �M) at 5, 13 and 25 C. Data are means ± SEM (n = 5). Points with

ifferent letters are statistically different from each other (two-way ANOVA withukey’s HSD, P < 0.05).

. Results

.1. Mitochondrial respiration

The effects of Cd on mitochondrial respiration were different athe three temperatures tested as clearly demonstrated by signifi-ant two-way interactions (Table 1). As well, temperature and Cdndividually significantly altered all of the mitochondrial respira-ion indices except that thermal sensitivity (Q10 coefficient) of state

was not affected by Cd.Temperature alone greatly stimulated (F2,72 = 671, P < 0.0001)

tate 3 respiration resulting in an overall 2.8-fold increase in res-iration between 5 and 25 ◦C (Fig. 1a) in the controls. In contrast,d dose-dependently inhibited (F5,72 = 222, P < 0.0001) state 3 res-iration at all the temperatures, resulting in 3-, 2.5- and 3-foldeductions in respiration rates relative to the corresponding con-

◦

rols in the 100 �M Cd exposure for 5, 13 and 25 C, respectivelyFig. 1a). Overall, a significant interaction (F10,72 = 18, P < 0.0001)etween Cd and temperature on state 3 respiration was observed.he temperature sensitivity of state 3 respiration (Fig. 1b) was

Fig. 3. Interactive effects of temperature and Cd on RCR. Mitochondria isolated fromeach fish were exposed to Cd (0, 5, 10, 20, 50 and 100 �M) at 5, 13 and 25 ◦C. Dataare means ± SEM (n = 5). Points with different letters are statistically different fromeach other (two-way ANOVA with Tukey’s HSD, P < 0.05).

J.O. Onukwufor et al. / Aquatic Toxicology 158 (2015) 75–87 79

F rrelati 25 ◦C.f

s(naw

n2Cti1iIst5sut

ig. 4. Interactive effects of temperature and Cd on (a) metal accumulation, and cosolated from each fish were exposed to Cd (0, 5, 10, 20, 50 and 100 �M) at 5, 13 androm each other (two-way ANOVA with Tukey’s HSD, P < 0.05).

ignificantly higher at the low (5–13 ◦C) compared with the high13–25 ◦C) temperature range (F1,48 = 558, P < 0.0001). There waso significant effect of Cd on state 3 Q10 (F5,48 = 1.08, P = 0.3847) but

significant temperature and Cd interaction (F5,48 = 5.6, P < 0.001)as evident.

Akin to state 3, the resting (state 4) respiration increased sig-ificantly (F2,72 = 2455, P < 0.0001) with temperature, attaining a.3-fold increase between 5 and 25 ◦C in the controls (Fig. 2a).admium (F5,72 = 114, P < 0.0001) acted in synergy with tempera-ure to further stimulate state 4 respiration (Fig. 2a) culminatingn a 3.6-fold increase in state 4 between 5 and 25 ◦C for the00 �M Cd exposure, a finding that is consistent with the significant

nteraction (F10,72 = 53, P < 0.0001) between Cd and temperature.n contrast with state 3 respiration, the control Q10 values fortate 4 respiration (Fig. 2b) increased (F1,48 = 2238, P < 0.0001) withemperature from 1.1 to 1.8 between the temperature ranges

–13 and 13–25 ◦C, respectively. While Cd exposure did not affecttate 4 thermal sensitivity over the 5–13 ◦C range, the Q10 val-es were increased (F5,48 = 47, P < 0.0001) by Cd doses >20 �M overhe 13–25 ◦C range. A significant (F5,48 = 21, P < 0.0001) interaction

ion of state 3 and Cd accumulation at (b) 5 ◦C, (c) 13 ◦C and (d) 25 ◦C. Mitochondria Data are means ± SEM (n = 5). Points with different letters are statistically different

between temperature and Cd on state 4 thermal sensitivity wasobserved. Similar to state 4 respiration, state 4ol (Fig. S1), a measureof proton leak, was highly stimulated by temperature (F2,72 = 1826,P < 0.0001) and Cd exposure (F5,72 = 104, P < 0.0001) intensified thisleak as verified by the significant two-way interaction (F10,72 = 46,P < 0.0001).

The P/O ratio, which indicates mitochondrial phosphorylationefficiency, was increased (F2,48 = 76, P < 0.0001) by temperature(Table 2) and reduced (F3,48 = 22, P < 0.0001) by Cd exposure. Over-all, a significant (F6,48 = 9, P < 0.0001) two-way interaction betweenCd and temperature on P/O ratio was observed. Note that P/O ratiosfor Cd doses >20 �M were not calculated because the transition tostate 4 could not be unambiguously estimated.

The mitochondrial coupling efficiency (as estimated by the RCR)was significantly influenced (F2,72 = 657, P < 0.0001) by temperature(Fig. 3) and was highest at 13 ◦C for all Cd concentrations. Exposure

to Cd decreased (F5,72 = 919, P < 0.0001) the RCR at all temperatures,with mitochondria tested at high temperature (25 ◦C) showing thegreatest reduction. As well, the interaction between temperatureand Cd on RCR was significant (F10,72 = 24, P < 0.0001).

80 J.O. Onukwufor et al. / Aquatic To

Table 2Effects of Cd and temperature on P/O ratio in rainbow trout liver mitochondria.Mitochondria isolated from each fish were exposed to Cd (0, 5, 10, 20, 50 and100 �M) at 5, 13 and 25 ◦C. Data are means ± SEM (n = 5). Points with different let-ters are statistically different from each other (two-way ANOVA with Tukey’s HSD,P < 0.05). NM indicates not measured.

Cadmium (�M) Temperature (◦C)

5 13 25

0 2.51 ± 0.07cd 3.42 ± 0.06a 3.61 ± 0.11a

5 2.54 ± 0.05cd 3.11 ± 0.07b 3.50 ± 0.14a

10 2.43 ± 0.12d 3.00 ± 0.08b 3.29 ± 0.07ab

20 2.42 ± 0.12d 2.94 ± 0.09bc 2.42 ± 0.02d

3

dCta

amplitude (Fig. 5c vs. d) of swelling. To test if the early con-

Fsa

50 NM NM NM100 NM NM NM

.2. Mitochondrial Cd accumulation

The amount of Cd accumulated by mitochondria increased dose-ependently (F = 357.75, P < 0.0001) with all exposures ≥20 �M

5,81d being significantly different from the controls (Fig. 4a). Impor-antly, elevated temperature enhanced (F2,81 = 7.85, P < 0.001) Cdccumulation as shown by the significant two-way interaction

15 oC

Time (s)

0 20 0 40 0 60 0 800 10 00 1200 1400 1600 1800 200 0

Abso

rban

ce54

0 nm

-0.0 8

-0.0 6

-0.0 4

-0.0 2

0.00

0.02

0.04a)

Ctl Ca 5 µµM Cd

Abso

rban

ce54

0 nm

-0.10

-0.08

-0.06

-0.04

-0.02

0.00c) d

Ctl Ca 5µµMCd 50µµMCd 100µµMC d

bb

b

a

b

ig. 5. Dose–response of Cd on mitochondrial volume changes. Mitochondrial suspensiowelling kinetics were monitored every 10 s for 30 min as 540 nm absorbance changes atre shown in (c) (15 ◦C) and (d) (25 ◦C). Points with different letters are statistically differ

xicology 158 (2015) 75–87

(F10,81 = 3.61, P < 0.001) between Cd and temperature. That theaccumulated Cd caused the mitochondria dysfunction observedwas confirmed by the strong inverse relationships between state3 and mitochondrial Cd concentration with R2 values of 0.93, 0.82,and 0.82 for 5, 13 and 25 ◦C, respectively (Fig. 4b–d).

3.3. Mitochondrial volume

Cadmium at concentrations ≥50 �M initially caused themitochondria to contract before swelling (Fig. 5a and b) withan overall significant treatment effect at both 15 ◦C (Fig. 5c:F4,20 = 11.51, P < 0.001) and 25 ◦C (Fig. 5d: F4,20 = 27.59, P < 0.0001).However, the amplitude of swelling achieved after 30 min wasnot different from that of the controls for all the Cd doses.As expected, the positive control (Ca, 200 �M) caused highlysignificant swelling that, unlike that caused by Cd, was notassociated with an initial contraction phase. Moreover, tem-perature had no effect on the pattern (Fig. 5a vs. b) or

traction caused by high Cd doses was due to a chemical orphysical interaction between Cd and buffer constituents, 100 �MCd was added to the swelling buffer alone and absorbance

b)

50 µµM Cd 100 µµM Cd

25 oC

Time (s)

0 200 400 600 800 1000 1200 14 00 16 00 18 00 2000-0. 08

-0. 06

-0. 04

-0. 02

0.00

0.02

0.04

-0.10

-0.08

-0.06

-0.04

-0.02

0.00)

Ctl Ca 5µµMC d 50µµMCd 10 0µµMCd

bc

a

c

bc b

ns were exposed to Cd (0, 5, 50 and 100 �M) and 200 �M Ca (positive control) and (a) 15 ◦C and (b) 25 ◦C. The means ± SEM (n = 5) amplitude of swelling after 30 minent from each other (one-way ANOVA with Tukey’s HSD, P < 0.05).

J.O. Onukwufor et al. / Aquatic Toxicology 158 (2015) 75–87 81

15 oC

Time (s)

0 200 400 600 800 1000 1200 1400 1600 1800 2000

Abso

rban

ce54

0 nm

-0.12

-0.10

-0.08

-0.06

-0.04

-0.02

0.00

0.02

0.0425 oC

Time (s)

0 200 40 0 60 0 800 100 0 120 0 1400 16 00 180 0 200 0-0.12

-0.10

-0.08

-0.06

-0.04

-0.02

0.00

0.02

0.04a) b)

Ctl Ca 5 µµM Cd 50 µµM Cd 100 µµM Cd

Abso

rban

ce54

0 nm

-0.12

-0.10

-0.08

-0.06

-0.04

-0.02

0.00

-0.12

-0.10

-0.08

-0.06

-0.04

-0.02

0.00c) d)

Ctl Ca 5µµMCd 50µµMC d 100µµMC dCtl Ca 5µµMCd 50µµMC d 10 0µµMCd

c

a

bbc

d

a

b

c

bc bc

Fig. 6. The effect of temperature shock (4 → 15 or 25 ◦C) on Cd-induced mitochondrial volume changes. Mitochondrial suspensions were exposed to Cd (0, 5, 50 and 100 �M)and 200 �M Ca (positive control) and swelling kinetics were monitored every 10 s for 30 min as 540 nm absorbance changes at (a) 15 ◦C and (b) 25 ◦C. The means ± SEM( ointsw

m(cPttctsre

asWsCm2mutP

n = 5) amplitude of swelling after 30 min are shown in (c) (15 ◦C) and (d) (25 ◦C). Pith Tukey’s HSD, P < 0.05).

onitored for 30 min; there were no changes in absorbanceFig. S2). Finally, temperature shock resulted in greater volumehanges at both 15 (F4,20 = 58.04, P < 0.0001) and 25 (F4,20 = 38.47,

< 0.0001) ◦C (Fig. 6) compared with mitochondria equilibrated toest temperature prior to the swelling assay (Fig. 5). Moreover,emperature-shocked mitochondria exhibited complex volumehanges with two phases of contraction and swelling. Interestingly,he low dose (5 �M) of Cd reduced the amplitude of spontaneouswelling after temperature shock. Lastly, while Ca-induced swellingeached steady in 4–5 min swelling evoked by Cd did not level outven after 30 min.

The prediction that Cd-induced mitochondrial volume changesre mediated by mitoKATP, MPTP and MCU were tested usingpecific modulators of these mitochondrial membrane pathways.

e found that the mitoKATP blocker, 5-HD, reduced Ca-inducedwelling but had no effect on volume changes associated withd (Fig. 7a and b). However, a significant treatment effect onitochondrial volume at 15 (Fig. 7c: F5,24 = 228.29, P < 0.0001) and

5 (Fig. 7d: F5,24 = 29.8, P < 0.0001) ◦C was observed. Diazoxide, a

itoKATP agonist, had no effect on both Ca- and Cd-induced vol-

me changes (Fig. 8a–d) although there were overall significantreatment effects on the volume changes both at 15 (F5,24 = 46.78,

< 0.0001) and 25 (F5,24 = 33.40, P < 0.0001) ◦C (Fig. 8c and d). With

with different letters are statistically different from each other (one-way ANOVA

regard to the role of MPTP, CsA, an inhibitor of MPTP, reducedCa-induced swelling but did not alter Cd-induced volume changes(Fig. 9a–d). A significant treatment effect was observed for testsdone at both 15 (F5,24 = 31.98, P < 0.0001) and 25 (F5,24 = 18.05,P < 0.0001) ◦C (Fig. 9c and d). Ruthenium red, an MCU blocker, com-pletely blocked Ca-induced swelling and abolished the Cd-inducedcontraction and spontaneous (control) swelling at both 15 and 25 ◦C(Fig. 10a–d). Significant treatment effects were observed at both15 ◦C (F5,24 = 74.58, P < 0.0001) and 25 ◦C (F5,24 = 46.94, P < 0.0001).Lastly, ruthenium red (Fig. 10a), and to a lesser extent CsA (Fig. 9a),altered the kinetics of Cd-induced swelling at 15 ◦C.

4. Discussion

The co-occurrence of fluctuating temperatures and elevatedmetals concentrations in aquatic systems calls for increased under-standing of their combined effects on the physiology of residentorganisms in order to more accurately predict the environmentalimpacts. To identify the potential interactive effects of Cd and tem-

perature on mitochondrial function and volume we assessed theeffects of the metal at three temperatures. We demonstrated thatall mitochondrial respiration indices assessed except the thermalsensitivity of state 3 were significantly modulated by temperature

82 J.O. Onukwufor et al. / Aquatic Toxicology 158 (2015) 75–87

15 oC

Time (s)

0 200 400 600 800 100 0 1200 14 00 160 0 1800 20 00

Abso

rban

ce54

0 nm

-0.1 0

-0.0 8

-0.0 6

-0.0 4

-0.02

0.00

0.02

25 oC

Time (s)

0 200 400 60 0 800 100 0 1200 14 00 160 0 1800 20 00-0.10

-0.08

-0.06

-0.04

-0.02

0.00

0.02

5-HD

a) b)

CtlCtl + 5-HD

CaCa + 5-HD

100 μμM Cd100 μμM Cd + 5-HD

Ctl Ctl + 5-H D Ca Ca + 5-H D Cd Cd + 5-H D-0.10

-0.08

-0.06

-0.04

-0.02

0.00

Ctl Ctl + 5-H D Ca Ca + 5-H D Cd Cd + 5- HD

Abso

rban

ce54

0 nm

afte

r 30

min

-0.10

-0.08

-0.06

-0.04

-0.02

0.00 )d)c

c c

b

c c

a

b b

aa

bb

Fig. 7. The effect of 5-hydroxydecanoate on Cd-induced mitochondrial volume changes. Mitochondrial suspensions were exposed to Cd (0 and 100 �M) and 200 �M Ca ands ges ata differ

aIogtm

crima22dGcIsi(2l

welling kinetics were monitored every 10 s for 30 min as 540 nm absorbance chanre shown in (c) (15 ◦C) and (d) (25 ◦C). Points with different letters are statistically

nd Cd exposure with significant two-way interactions (Table 1).mportantly, the inhibitory effect of Cd on OXPHOS and its effectsn volume (Figs. 5 and 6) were greater at high temperature sug-esting that temperature increases as occurs naturally and/or dueo global climate change may make fish energy generating systems

ore vulnerable to metals such as Cd.Individually temperature had stimulatory effects on mito-

hondrial respiration, increasing the phosphorylating (state 3)espiration by approximately 3× between 5 and 25 ◦C. This findings consistent with several other studies in a wide range endother-

ic and ectothermic animal species (Willis et al., 2000; Bouchardnd Guderley, 2003; Birkedal and Gesser, 2003; Fangue et al.,009; Lemieux et al., 2010; Zukiene et al., 2010; Schulte et al.,011). At low temperatures the aerobic capacity of the mitochon-ria is believed to be limited by low substrate oxidation (Blier anduderley, 1993) and diffusion (Dunn, 1988) rates that are in partaused by changes in membrane properties (Kraffe et al., 2007).n converse, raising the temperature increases the rates of sub-trate oxidation because of stimulation of activities of enzymes,

ncreased diffusion rates and decreased substrate binding affinitiesBlier and Guderley, 1993; Guderley and Johnston, 1996; Somero,011), resulting in high respiration rate. Contrasting the stimu-

atory effect of temperature, Cd dose-dependently reduced state

(a) 15 ◦C and (b) 25 ◦C. The means ± SEM (n = 5) amplitude of swelling after 30 minent from each other (one-way ANOVA with Tukey’s HSD, P < 0.05).

3 respiration at all temperatures (Fig. 1a) which is in agreementwith several previous studies (Kesseler and Brand, 1994b; Adieleet al., 2010; Kurochkin et al., 2011; Onukwufor et al., 2014).The mechanisms through which Cd inhibits state 3 respirationinclude direct impairment of the activity of ETC enzymes andsubstrate transporters (Rikans and Yamano, 2000; Wang et al.,2004; Kurochkin et al., 2011), inhibition of substrate oxidation(Ivanina et al., 2008), increased mitochondrial membrane perme-ability (Belyaeva and Korotkov, 2003), and uncoupling of OXPHOS(Belyaeva et al., 2001; Adiele et al., 2010), leading to the overallinhibition of OXPHOS (Dorta et al., 2003; Wang et al., 2004). Thecombined exposure showed significant interaction between tem-perature and Cd wherein thermal stress (5 and 25 ◦C) exacerbatedthe inhibitory effect of Cd on state 3 mitochondrial respiration.This suggests that both elevation and decrease in environmentaltemperature would lead to greater disturbances of OXPHOS in fish.

The increase in state 4 respiration (Fig. 2a) observed on rais-ing the temperature from 5 to 25 ◦C is consistent with severalother studies (Abele et al., 2002; Chamberlin, 2004; Sokolova, 2004;

Fangue et al., 2009) and was largely due to increase in state 4ol(proton leak). It is likely that high temperature stimulates mech-anisms that mediate proton leak such as the adenine nucleotidetranslocase, uncoupling proteins and other IMM proteins (Jastroch

J.O. Onukwufor et al. / Aquatic Toxicology 158 (2015) 75–87 83

15 oC

Time (s)

0 20 0 40 0 600 800 1000 1200 1400 160 0 180 0 200 0

Abso

rban

ce54

0 nm

-0.10

-0.08

-0.06

-0.04

-0.02

0.00

0.02

0.04

25 oC

Time (s)

0 200 400 600 800 1000 1200 1400 1600 1800 2000-0.10

-0.08

-0.06

-0.04

-0.02

0.00

0.02

0.04

Diazoxide (Diaz)

a) b)

CtlCtl + Diaz Ca + Diaz

100 µµM CdCa100 µµM Cd + Diaz

Ctl Ctl + Diaz Ca Ca + Diaz Cd Cd + Diaz-0.10

-0.08

-0.06

-0.04

-0.02

0.00

Ctl Ctl + Diaz Ca Ca + Diaz Cd Cd + Diaz

Abso

rban

ce54

0 nm

afte

r 30

min

-0.10

-0.08

-0.06

-0.04

-0.02

0.00c) d)

bb

a

a

bb

b b

a

a

b b

F chondk 5 ◦C ai om ea

epflTt4stR

taCvWcreS(otWtpC

ig. 8. The effect of diazoxide on Cd-induced mitochondrial volume changes. Mitoinetics were monitored every 10 s for 30 min as 540 nm absorbance changes at (a) 1n (c) (15 ◦C) and (d) (25 ◦C). Points with different letters are statistically different fr

t al., 2010). Alternatively, temperature-induced increase in IMMermeability (Dahlhoff and Somero, 1993) would elevate inwardux of protons (Echtay et al., 2002; Goglia and Skulachev, 2003;albot et al., 2004). Interestingly, the combined exposure showedhat temperature and Cd acted cooperatively in stimulating state/proton leak suggesting that concurrent exposure to thermaltress and Cd would reduce mitochondrial efficiency. In additiono reducing ATP synthesis, high proton leak may lead to increasedOS production in inhibited mitochondria (Brookes, 2005).

The Q10 values measured in the present study indicate thathe thermal sensitivity of state 3 respiration (Fig. 1b) was highert low temperature which is in line with an earlier study byhamberlin (2004). Interestingly, Cd exposure increased the Q10alues of state 3 respiration over the low temperature range.hile the mechanisms underlying the high state 3 temperature

oefficients at low temperature and their enhancement by Cdemain to be determined, this finding implies that mitochondriaxhibit exaggerated changes in OXPHOS when faced with Cd stress.urprisingly, unlike the maximal respiration, state 4 Q10 valuesFig. 2b) were higher at high temperature and increased furthern exposure to Cd indicating a synergistic interaction of tempera-ure and Cd on basal mitochondrial respiration thermal sensitivity.

hile ours is the first study to report that Cd increases state 4hermal sensitivity, high Q10 values for this metric at the high tem-erature have been observed in earlier studies (Hulbert et al., 2002;hamberlin, 2004). Because state 4 respiration was predominantly

rial suspensions were exposed to Cd (0 and 100 �M) and 200 �M Ca and swellingnd (b) 25 ◦C. The means ± SEM (n = 5) amplitude of swelling after 30 min are shownch other (one-way ANOVA with Tukey’s HSD, P < 0.05).

proton leak (Fig. S1) the enhancement of sensitivity of state 4 totemperature by Cd indicates decreased mitochondrial efficiencywhich is aligned with the lower RCR (Fig. 3) and P/O ratio (Table 2) inthe high temperature–Cd exposure. Conversely, increased protonleak may serve a protective function by reducing ROS generationand associated oxidative damage under these conditions. Similarto our findings, temperature was shown to increase the sensi-tivity of oyster gill mitochondria to Cd (Sokolova, 2004). Overall,the inhibition of state 3 coupled with preferential stimulation of4 culminated in greatly reduced RCR in the high temperature–Cdexposure indicative of highly compromised OXPHOS.

The prediction that the greater mitochondrial dysfunctionobserved following Cd exposure at high temperature results fromincreased Cd accumulation was confirmed (Fig. 4). Notably, therewere strong correlations between state 3 respiration and log mito-chondrial Cd concentration at all temperatures indicating thatlow burdens of Cd imposed big reductions in respiration whilehigh burdens caused small reductions. Though the mechanismsof enhancement of mitochondrial Cd uptake at high temperaturewere not explored, it is possible that the IMM exhibited increasedleakiness (Dahlhoff and Somero, 1993; Abele et al., 2002), therebyallowing greater influx of Cd. An alternative explanation for the

increased Cd accumulation at high temperature is increased activ-ity of mitochondrial transporters such as the MCU which ourstudy and others (Lee et al., 2005a; Adiele et al., 2012) haveshown to be involved in Cd uptake by the mitochondria. Because

84 J.O. Onukwufor et al. / Aquatic Toxicology 158 (2015) 75–87

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Tim e (s )

0 20 0 40 0 60 0 80 0 10 00 120 0 140 0 160 0 180 0 200 0

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0 nm

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0.00

0.02

0.0425 oC

Tim e (s)

0 200 400 600 800 1000 1200 1400 1600 1800 2000-0.08

-0.06

-0.04

-0.02

0.00

0.02

0.04

Cyclosporin A (CsA)

a) b)

CtlCtl + CSA

CaCa + CSA

100 µµM Cd100 µµM Cd + CSA

Ctl Ctl + CS A Ca Ca + C SA Cd Cd + CSA-0. 08

-0. 06

-0. 04

-0.02

0.00

Ctl Ctl + CSA Ca Ca + CSA Cd Cd + C SA

Abso

rban

ce54

0 nm

afte

r 30

min

-0.08

-0.06

-0.04

-0.02

0.00c) d)

c

c

a

b

c c

c

c

ab

b

c cb

F tochok 5 ◦C ai om ea

sr(Hp

sridomesttiawcsdvu2mtm

ig. 9. The effect of cyclosporin A on Cd-induced mitochondrial volume changes. Miinetics were monitored every 10 s for 30 min as 540 nm absorbance changes at (a) 1n (c) (15 ◦C) and (d) (25 ◦C). Points with different letters are statistically different fr

imilar enhancement of Cd accumulation at high temperature waseported at the organism level in organisms of different classesGoncalves et al., 1988; Tessier et al., 1994; Bervoets et al., 1996;eugens et al., 2003), it appears that the augmenting effect of tem-erature on Cd accumulation is a generalized phenomenon.

The mitochondria functional integrity is guaranteed by theelective permeability of the IMM which regulates the flow of mate-ials between the matrix and the cytosol/intermembrane space and,mportantly, allows the generation of a protonmotive force thatrives ATP synthesis (Mitchell, 1966). Despite the importance ofsmotic movement of water into and out of the mitochondria foritochondrial function (Nowikovsky et al., 2009), the interactive

ffects of metals (e.g., Cd) and thermal stress on volume homeosta-is of these organelles have not been investigated. We thereforeested the idea that the deleterious effects of Cd and adverseemperature on mitochondria result from volume changes. By mon-toring mitochondrial volume changes (i.e., changes in absorbancet 540 nm) over time as an indicator of changes in IMM permeabilitye showed that Cd at doses ≥50 �M (Fig. 5) evokes early mito-

hondrial contraction before inducing swelling. Cadmium-inducedwelling-contraction has been reported in mammalian mitochon-ria by Lee et al. (2005a,b) who, similar to our study, monitoredolume changes kinetically. A study that reported endpoint vol-me measurements detected only contraction in oyster (Sokolova,

004) and suggested that oyster mitochondria do not undergo per-eability transition and associated swelling. The author speculated

hat oyster mitochondria retain sufficient proton pump activity toitigate depolarization and swelling when exposed to Cd. More

ndrial suspensions were exposed to Cd (0 and 100 �M) and 200 �M Ca and swellingnd (b) 25 ◦C. The means ± SEM (n = 5) amplitude of swelling after 30 min are shownch other (one-way ANOVA with Tukey’s HSD, P < 0.05).

recently, Adiele et al. (2012) observed mild swelling in an endpointassay using rainbow trout liver mitochondria and argued that livermitochondria in this species are recalcitrant to swelling. Our study,in particular the effect of Ca, indicates that rainbow trout livermitochondria are capable of swelling, highlighting the importanceof kinetic measurement of mitochondrial volume changes.

Mitochondria behave like osmometers capable of swelling andcontraction due to water movement that accompanies the nettransport of osmotically active solutes into and out of the matrix ofthese organelles (Beavis et al., 1985; Kaasik et al., 2007). Regardingthe contraction observed in the early phase of Cd exposure, the pos-sibility that the change in absorbance was due to Cd or complexesformed by reaction of Cd with components of the swelling bufferwas ruled out (Fig. S2). Secondly, because our assay was done inthe absence of added ADP, mitochondrial conformational changescaused by ADP and ATP that increase light scatter without alteringmatrix volume (Das et al., 2003 and references therein) could alsonot have been a causal factor. Furthermore, the possibility of anincrease in refractive index due to Cd complexation with phosphatein the mitochondrial matrix that can be interpreted as contrac-tion as observed following Ca exposure of brine shrimp, Artemiafranciscana, mitochondria (Menze et al., 2005; Holman and Hand,2009), was ruled out because the contraction we observed was tran-sient, whereas the formation of calcium phosphate in brine shrimp

mitochondria was a permanent monotonic phenomenon. There-fore the mitochondrial contraction observed here was likely due tospecific effects of Cd on the mechanisms or structures that reg-ulate solute and water transport in these organelles. A possible

J.O. Onukwufor et al. / Aquatic Toxicology 158 (2015) 75–87 85

15 oC

Time (s)

0 200 400 600 80 0 1000 1200 140 0 1600 1800 2000

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0 nm

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0.00

0.0225 oC

Time (s )

0 200 400 600 800 1000 1200 1400 160 0 1800 2000-0.10

-0.08

-0.06

-0.04

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0.00

0.02

Ruthenium red (RR)

)b)a

CtlCtl + RR

100 µµM Cd100 µµM Cd + RR

CaCa + RR

Ctl Ctl + RR Ca Ca + RR Cd Cd + RR-0.10

-0.08

-0.06

-0.04

-0.02

0.00

Ctl Ctl + RR Ca Ca + RR Cd Cd + RR

Abso

rban

ce54

0 nm

afte

r 30

min

-0.10

-0.08

-0.06

-0.04

-0.02

0.00

c) d)

be

d

a

e

bdebd

bd

d

a

cb

cc

F s. Mits ges ata differ

ecmtcgots1aalwci

dcitesd

ig. 10. The effect of ruthenium red on Cd-induced mitochondrial volume changewelling kinetics were monitored every 10 s for 30 min as 540 nm absorbance chanre shown in (c) (15 ◦C) and (d) (25 ◦C). Points with different letters are statistically

xplanation is that Cd at high doses initially activated mito-hondrial K+/H+ exchanger leading not only to the dissipation ofembrane potential but also contraction due to loss of water from

he matrix associated with K+ efflux. With time, however, matrixontraction would block the K+/H+ exchange creating an osmoticradient that drives K+ back into the matrix causing progressivesmotic mitochondrial swelling which may ultimately cause rup-ure of the outer membrane with leakage of the intermembranepace contents into the cytosol (Zoratti and Szabo, 1995; Bernardi,999; Halestrap et al., 2002). It is also possible that Cd activatesquaporins, channels that regulate the osmotic movement of watercross IMM (Ferri et al., 2003), thus increasing influx of watereading to swelling. Indeed, blocking mitochondrial aquaporins

ith AgNO3 abolished Cd-induced swelling (Lee et al., 2005a), aompelling indication that these channels are involved in the Cd-nduced mitochondrial volume changes.

Our mechanistic analysis revealed that contrary to our pre-iction, Cd-induced rainbow trout liver mitochondrial volumehanges are not mediated by MPTP. Specifically, CsA, an MPTPnhibitor, did not stop or reduce Cd-induced swelling (Fig. 9). Note

hat the induction of MPTP occurs in these mitochondria (Adielet al., 2012) and was confirmed here by the finding that Ca-inducedwelling was CsA sensitive. The inability of CsA to block mitochon-rial swelling is not unique to our study having been observed in

ochondrial suspensions were exposed to Cd (0 and 100 �M) and 200 �M Ca and (a) 15 ◦C and (b) 25 ◦C. The means ± SEM (n = 5) amplitude of swelling after 30 minent from each other (one-way ANOVA with Tukey’s HSD, P < 0.05).

previous studies using Cd (Lee et al., 2005b), Hg2+ (Eliseev et al.,2002) and long-chain fatty acid (Sconfeld et al., 2000) as inducers.However, Cd-induced volume changes were reduced by ruthe-nium red (Fig. 10), an inhibitor of the MCU, indicating that Cdentry through this channel is an important requirement for theobserved volume changes. Lastly, neither diazoxide (Fig. 8) nor 5-HD (Fig. 7) had effect on Cd-induced volume changes, suggestingnon-involvement of mitoKATP despite the importance of K+ fluxes inthe regulation of mitochondrial volume (Garlid et al., 1996; Jabureket al., 1998; Lee et al., 2005b). Note, however, that mitoKATP havenot always been associated with mitochondrial volume regulation(Das et al., 2003).

The mitochondrial volume changes observed in our study, witha clear early contraction followed by two phases of swellingwere highly influenced by temperature. While mitochondriaequilibrated to test temperature showed moderate contractionand swelling (Fig. 5), those tested after temperature shock(abrupt transfer from 4 → 15 or 25 ◦C) had complex patternsof contraction and swelling with higher amplitudes (Fig. 6).The complex swelling-contraction pattern is possibly associated

with temperature-induced changes on mitochondrial membranecharacteristics (Connell, 1961; Richardson and Tappel, 1962;Somero, 2011). Interestingly, low doses of Cd prevented spon-taneous swelling in both equilibrated and temperature-shocked

8 tic To

mmcmi

5

tWalittIioCstu

A

i(

A

fj

R

A

A

A

B

B

B

B

B

B

B

B

B

6 J.O. Onukwufor et al. / Aqua

itochondria, suggesting that at low doses Cd influences theechanisms of solute and water movement across the inner mito-

hondrial membrane. Additional studies are required to unveil theechanisms by which low Cd doses inhibit spontaneous swelling

n isolated mitochondria.

. Conclusions

We have shown that temperature and Cd exposure interac-ively impair mitochondrial function and dysregulate their volume.

hereas individually temperature stimulated both the maximalnd leak respirations, Cd inhibited the former while stimulating theatter, leading to severe uncoupling and exacerbation of functionalmpairment in the combined exposure. Interestingly, Cd increasedhe thermal sensitivity (Q10 values) of maximal respiration at lowemperature and that of basal respiration at high temperature.mportantly, we show that the aggravation of Cd-induced mitotox-city at high temperature was in part due to increased accumulationf the metal in the organelles. Additionally, our data suggest thatd alters mitochondrial permeability leading to contraction andwelling that is aggravated by temperature change. It appearshat Cd-imposed mitochondrial volume alterations require metalptake via the MCU.

cknowledgement

This study was funded by Natural Sciences and Engineer-ng Research Council of Canada Discovery Grant awards to CK#311929-2011) and DS (#104-2008).

ppendix A. Supplementary data

Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/.aquatox.2014.11.005.

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