Ecotoxicology (2014) 23:1846–1853 DOI 10.1007/s10646-014-1312-9
Perchlorate-induced oxidative stress in isolated liver mitochondria Xiaohu Zhao • Peijiang Zhou • Xiu Chen Xi Li • Ling Ding
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Accepted: 9 August 2014 / Published online: 20 August 2014 Ó Springer Science+Business Media New York 2014
Abstract As a new threat to environment all through the world, perchlorate (ClO4-) was predominantly a thyrotoxin, and its toxic manifestations in non-thyroid were also documented. To date, little is known about the effect of ClO4- on cell and organelle. To reveal the toxicity of ClO4- on living organism in-depth, mitochondria isolated from liver of Carassius auratus were incubated with different concentrations of ClO4-. The results demonstrated that ClO4--induced mitochondrial oxidative stress, and subsequently caused a gradual opening of permeability transition pore leading to mitochondrial swelling and lipid peroxidative membrane damage. ClO4- has a conspicuous inhibition of electron transport chain activity which largely correlated to complexes I and IV. The investigations clearly demonstrated the oxidative stress of ClO4- in mitochondria, may well reveal cytotoxic effects in vitro that merit further investigation.
X. Zhao P. Zhou (&) X. Chen X. Li L. Ding Hubei Biomass-Resource Chemistry and Environmental Biotechnology Key Laboratory, College of Resources and Environmental Sciences, Wuhan University, Wuhan 430072, Hubei, China e-mail: [email protected]; [email protected] X. Zhao e-mail: [email protected] X. Chen e-mail: [email protected] X. Li e-mail: [email protected] L. Ding e-mail: [email protected] X. Zhao Micro-element Research Center, Huazhong Agricultural University, Wuhan 430070, China
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Keywords Perchlorate Oxidative stress Liver Mitochondria
Introduction Perchlorate (ClO4-) and its salts are powerful oxidant that are widely used in rocket fuels, propellants, explosives (Attanasio et al. 2011). Because of the relatively high solubility in water, ClO4- would be produced once perchlorate salts released into aqueous systems. Due to the rapid mobility, stability, and nonreactivity, ClO4- in environment can persist for years to decades. Studies indicated that perchlorate is also found in a variety of foods and food crops (Sanchez et al. 2006; Murray et al. 2008), the milk from cows (Sanchez et al. 2008), and human breast milk (Pearce et al. 2007). Perchlorate is regarded as a new emerging persistent inorganic contaminant (Can˜as et al. 2006), and a new threat to environment all through the world. Due to potential toxicity, perchlorate has become serious threat to human health. To determine the toxic effects of perchlorate, experiments in vivo and pathological studies were used frequently. Studies have reported that perchlorate in the human body interferes with the uptake of iodine into the thyroid and may interfere with the development of the skeletal system and the central nervous system of infants (Borjan et al. 2011). In addition, treatment with high concentration of ClO4- significantly influenced the growth of preantral follicles and antral follicles, as well as other organisms (Bradford et al. 2005). However, based on its toxicity on living organism, there are relatively few studies on toxic effects of ClO4- in vitro, or has not been investigated in-depth. Moreover, little is known about the mechanism by which ClO4- act on cellular level or molecular level.
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Due to ClO4- in aquatic systems can persist for many years, fish in contaminated water may be affected by ClO4-. The liver is known to be the most important target organ involved in metabolism and detoxification for xenobiotics in vertebrates. The mitochondria is an important organelle which, in addition to ATP synthesis, participate in cytosolic calcium homeostasis regulation and cell signaling (Zhang et al. 2008). It also participates in important regulative processes like apoptosis. Hence, the maintenance of mitochondrial activity is the key aspect of cellualr survival particularly under stressed conditions (Parihar et al. 2009). The freshwater fish Carassius auratus, commonly found in China and widely used in aquatic toxicology, was chosen as the test organism (Feng et al. 2013). Previous research in our laboratory revealed that ClO4influenced the metabolic activity of mitochondria and cell (Zhao et al. 2011), and ClO4- increased ROS and MDA in hepatocytes isolated from Carassius auratus. To investigate mitochondrial functional alternation related to ClO4-induced oxidative stress, mitochondria isolated from liver of Carassius auratus were incubated with different concentrations of ClO4-. The present work is the first attempt to explore the influence of ClO4- stress on oxidative stress associated mitochondria, which would contribute to a better understanding of the toxic effect of ClO4- to aquatic organisms.
Materials and methods Chemicals Analytical-reagent grade NaClO4 was obtained from Tianjin Kermel Chemical Reagent Co., Ltd (Tianjin, People’s Republic of China). The mitochondria isolation medium A contained 250 mM sucrose, 50 mM Tris–HCl, 1 mM Na2EDTA, 0.1 % BSA, 1 mM 2-mercaptoethanol, 10 mM Tris–HCl, pH7.5 and isolation medium B contained 250 mM sucrose, 50 mM Tris–HCl, pH 7.5. The lipid peroxidation (MDA) detection kit was purchased from Nanjing jiancheng Bioengineering Institute (Nanjing, China). All other reagents were of the purest grade available and purchased from Sigma unless indicated otherwise. Fish sampling The present study was conducted in accordance with the Guiding Principles in the Use of Animals in Toxicology. Carassius auratus (0.2–0.3 kg each) were provided by Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China). They were kept in 300 L fiber-glass tanks with aerated, dechlorinated water (20 ± 2 °C, 12 h
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light:12 h dark cycles) for about 10 days before hepatocytes isolation. Fish was sacrificed by severing their spinal cord and the liver was removed immediately for the isolation of mitochondria. Isolation of mitochondria The mitochondria were isolated according to the methods of Liu et al. (2000) and Zhu et al. (2006), with a slight modify. First, removed liver tissue from Carassius auratus, weighed and rinsed with cold sterilized isolation mediun A, and then minced, homogenized, and centrifuged at 1,000 rpm for 15 min. The clear supernatant was centrifuged again for another 15 min at 1,000 rpm. Next, the pellet was suspended in isolation medium A and centrifuged at 15,000 rpm for 10 min in a new tube. The resulting pellet was washed twice with isolation medium B, centrifuged for 30 min at 15,000 rpm. The purified mitochondria were suspended again in isolation medium B. All of the above operations were performed aseptically at 0–4 °C. We maintained the equal concentration of mitochondria protein at 1 mg protein mL-1 by adding the suspension medium. Protein content was measured by the method described in ‘‘Protein assay’’ section. Mitochondrial swelling assays Mitochondrial osmotic volume changes due to colloidal effects of solute flux into and out of the mitochondrial matrix was measured by monitoring the absorbance at 540 nm (Zhao et al. 2010). The assays were performed in 1 mL of incubation buffer (200 mM sucrose, 5 mM succinate, 2 lM rotenone, 10 lM EGTA, 3 mM HEPES, 1 mM KH2PO4, pH 7.4) with isolated mitochondria (1 mg protein), which were added into a 96-well microplate and incubated with ClO4- (0, 0.1, 1, 10, 100, 1,000 lM) at 30 °C. Absorbance at 540 nm was monitored using a microplate spectrophotometer (Sunrise, Tecan, Switzerland) at 2 min time intervals for 30 min. Here, the highest and non-effective doses of ClO4- were according to our previous study. Measurement of fluorescence polarization in isolated Mitochondria Fluorescence polarization was measured using 1,6-diphenyl-1,3,5-hexatriene (DPH) (Li et al. 2009). Briefly, after mitochondrial fractions (1 mg) were incubated with concentrations of ClO4- as mentioned above. Then washed and incubated in 1 mL 1 9 10-6 M DPH solution at pH 7.4, at 25 °C for 30 min. Fluorescence polarization was measured by a spectrofluorometer equipped with a polarization attachment at excitation 362 nm, and emission
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432 nm at 25 °C. The degree of fluorescence polarization (P) was calculated as: P ¼ ðIvv GIvh Þ=ðIvv þ GIvh Þ where Ivv and Ivh are the fluorescence intensities of emitted light with vertical excitation and vertical (horizontal) emission polarizer, respectively. The emission grating factor (G) corrected for the parallel diffraction anomalies introduced by the glass of the phototube was calculated as: G ¼ Ihv =Ihh Ihv (hv) is the intensity with horizontal excitation with horizontal (vertical) emission polarizer. Mitochondrial membrane fluidity (1/g) was calculated as: 1=g ¼ ð0:46 PÞ=2P Mitochondrial MDA assays Malondialdehyde (MDA) concentration was detected in isolated liver mitochondria using thiobarbituric acid reactive substances (TBARS) according to the protocol of MDA kit. In brief, 1 mL mitochondrial suspensions were incubated with concentrations of ClO4- as mentioned above, for 30 min at 25 °C. Then the supernatant fractions were collected and lipid peroxidation was estimated spectrophotometrically at 530 nm. The MDA concentration was expressed as nmol TBARS mg-1 protein. Measurement of ROS production in isolated mitochondria ROS production in isolated mitochondria was measured using a dichlorofluorescin diacetate (DCFH2-DA) fluorescence probe. Briefly, mitochondrial fractions (1 mg) were incubated with ClO4- (as mentioned above) for 30 min at 25 °C. After washing, mitochondrial suspensions were incubated with 20 lM of DCFH2-DA for 20 min at 30 °C. At the end of the incubation, mitochondria were washed with fresh buffer at 12 000 rpm for 15 min at 4 °C and resuspended. After, the fluorescence intensity was measured immediately using a microplate reader (Synergy HT, Bio-Tek Instruments, USA). Excitation and emission wavelengths were 490 and 530 nm, respectively (Zhang et al. 2008; Zhao et al. 2010). The ROS concentration was expressed as DCF fluorescence intensify.
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buffer (10 mM Tris–HCl, pH 7.4, 10 mM EDTA, 100 mM NaCl, 200 lg mL-1 proteinase K and 0.5 % SDS). Then, the lysate was extracted with phenol: chloroform: isoamyl alcohol (25:24:1) and centrifuged at 10 000 rpm for 5 min at 4 °C. Next, DNA was precipitated with 3 M sodium acetate and 100 % cold ethanol and deposited for 12 h at -20 °C. Then the precipitated was centrifuged at 12,000 rpm for 15 min at 4 °C. The DNA pellets were washed with 70 % ethanol, air-dried, and dissolved in Tris– EDTA buffer (10 mM Tris, 1 mM EDTA, pH 7.4). After that, 5 mg L-1 RNase A was added to each sample to remove RNA. Samples were electrophoresed on a 1.2 % agarose gel (containing 10 lg mL-1 ethidium bromide) for 1.2 h at 60 V. After eletrophoresis, the DNA ladder was visualized by Bioimaging System (Dong et al. 2009). Measurement of the mitochondrial respiratory chain complexes activity After incubated with various of ClO4- as mentioned above, washed, and mitochondrial fractions (1 mg) were freezed and thawed three times to fully expose the enzymes to substrates and achieve maximal activities. Complex I activity was measured in the presence of decylubiquinone and succinate as the rotenone sensitive decrease in NADH at 340 nm. Complex II activity was measured by the rate of 2,6-DCPIP reduction at 600 nm (Albayrak et al. 2003; Lemarie et al. 2009). Complex III activity was measured by following the initial antymicin A-sensitive rate of cytochrome c reduction at 550 nm in the presence of rotenone and decylubiquinone (Cela et al. 2010). Complex IV activity was assayed by following the decrease in absorbance due to the oxidation of previously reduced cytochrome c at 550 nm according to the method described by Rustin (1994). The activities of mitochondrial respiratory chain complexes were expressed as nmol/ min/mg protein. Protein assay Protein content was assayed according to Bradford (1976), using bovine serum albumin (Hyclone, USA) as a standard. Statistical analysis
DNA fragmentation assay The determination of DNA fragmentation pattern (DNA ladder) was carried out by agarose gel electrophoresis. After incubation as mentioned above, mitochondria were washed and then lysed for 1 h at 25 °C with 1 mL lysis
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Data presented are mean ± standard deviation (S.D.) of three different experiments. The statistical analysis was performed using one-way analysis of variance (ANOVA SPSS 18.0, Chicago, IL, USA) followed by Dunnett-t-test. The significance level was ascertained at P \ 0.05.
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Time (min) Fig. 1 The effects of ClO4- on mitochondrial swelling. Mitochondrial swelling was recorded by measuring the absorbance at 540 nm incubation with ClO4- (0, 0.1, 1, 10, 100, 1,000 lM) at 30 °C, at 2 min time intervals for 30 min. Each plot is representative of three independent measurements
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Fig. 3 Effect of ClO4- on MDA in isolated mitochondria. Data are reported as mean ± SD, N = 3. Asterisks indicate significant difference from control (**P \ 0.01)
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Results -
ClO4 induced isolated mitochondrial swelling The swelling of mitochondria reflected the opening of mitochondrial ion channels and membrane pores. As shown in Fig. 1, when in absence of ClO4-, mitochondria swells very slowly. However, the addition of ClO4- caused a concentration -dependent manner in mitochondrial swelling. A rapid swelling was not observed until the presence of 100–1,000 lM ClO4-. Effects of ClO4- on fluidity of isolated mitochondrial membrane The effect of increasing concentrations of ClO4- on DPH polarization of mitochondrial membrane was shown in
Fig. 2. No significant changes occurred in DPH polarization until 1,000 lM ClO4- treated. The result revealed that ClO4- can decrease the fluidity of mitochondrial membrane, but only in high concentration-treated.
Assessment of MDA in isolated mitochondria Levels of MDA, the last product of lipid breakdown caused by oxidative stress, were assessed in mitochondria from the control group. As shown in Fig. 3, MDA levels were significantly increased in the mitochondria treated with 1,000 lM ClO4- group, compared to the normal control. However, no significant difference was recorded between low-dose groups and the normal control group.
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Fig. 5 DNA fragmentation analysis of mitochondria by DNAagarose gel electrophoresis after exposure to various concentrations of ClO4- for 30 min. M marker, 1 control, 2 0.1 lM, 3 1 lM, 4 10 lM, 5 100 lM, 6 1,000 lM
Generation of ROS of isolated mitochondria Treatment of mitochondria with increasing concentrations of ClO4- resulted in a significant enhancement of the DCFrelated fluorescence indicating alterations of the intracellular redox state. As shown in Fig. 4, the DCF-fluorescence signal no or faintly changed after incubated with 0.1–100 lM ClO4-. However, the same result as polarization of mitochondrial membrane, when compared to the control group, the production of ROS was elevated only when treated with 1,000 lM ClO4-. Effects of ClO4- exposure on DNA fragmentation in isolated mitochondria The DNA of apoptotic cells presented as a regular ladder, which is typical of cells undergoing apoptosis. DNA fragmentation in isolated mitochondria was observed as shown in Fig. 5. Compare with the control, negligible DNA fragmentation produced when treated with 0.1–10 lM ClO4-. However, when mitochondria exposed to 100–1,000 lM ClO4-, DNA in mitochondria was cut into fragment. Effects of ClO4- exposure on the activities of mitochondrial complex To locate the inhibition sites in the mitochondrial respiratory chain, the effect of ClO4- was tested on the specific activities of complexes. The data in Fig. 6 show that there was a statistically decline of complex I and IV activity in
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Fig. 6 Effects of perchlorate on mitochondrial respiratory complex I, II, III and IV activities. Data are reported as mean ± SD, N = 3. Asterisks indicate significant difference from control (*P \ 0.05; **P \ 0.01)
isolated mitochondria when exposured to high concentrations of ClO4-, compared with the control. The activity of complex I reduced approximately 22.4 and 21.8 % when incubated with 100, 1,000 lM ClO4-, respectively. And the activity of complex IV reduced approximately 30.0, 37.7 and 48.4 % when incubated with 10, 100, 1,000 lM ClO4-, respectively. Interestingly, by an unknown mechanism, both the activity of complex II and complex III were unaffected by ClO4- at all the incubation-concentration tested.
Discussion Recently, the effects of ClO4- have been studied on diverse biological systems. Experiments in vivo showed that treatment with high concentration of ClO4- significantly influenced the growth of preantral follicles and antral follicles (Baldridge et al. 2004), as well as other organisms (Bradford et al. 2005). Moreover, growth indices of some organs/tissues were diminished by high concentration, but enhanced at low (Park et al. 2006). In previous work, we found that ClO4- influenced the metabolic activity of mitochondria and cell (Zhao et al. 2011). In addition, in the presence of high concentration of ClO4-, the primary cultured hepatocytes isolated from Carassius auratus started to shrink and became irregular in shape, displayed nuclear fragmentation, membrane blebbing, endoplasmic reticulum dilation, mitochondrial swelling or vacuolization, and the number of apoptotic cells and necrotic cells were significantly increased. Liver, which are particularly rich in mitochondria and that rely on aerobic ATP production are expected to be
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Fig. 7 Schematic view of the proposed mechanisms of ClO4--mediated ‘‘mitotoxicity’’. The action of ClO4- leading to collapse of MMP, consequent decrease of the ATP synthesis and the production of ROS are indicated in the model
more vulnerable to the action of toxin. Therefore, in the present study, to investigate mitochondrial functional alternation related to ClO4--induced oxidative stress and the cytotoxicity, mitochondria isolated from liver of Carassius auratus were incubated with different concentrations of ClO4- in this study. Mitochondria are involved in a large variety of cellular functions which are largely determined by the inner membrane potential. The change of the inner membrane potential may induce a series of reaction which cause the apoptosis. In addition, depolarization of mitochondria is an early sign of mitochondrial dysfunction and often recedes many other signs of cell injury (Muramatsu et al. 2007). In this study, the promotion of polarization of the inner membrane (Fig. 2), and mitochondrial swelling (Fig. 1) were observed, indicating change in membrane permeability. It is concluded that ClO4--induced mitochondrial permeability transition pore (mPTP) opening, which may caused destruction to hepatocytes. MDA is a terminal product of polyunsaturated fatty acid peroxidation, often used as a marker of oxidative damage associated with degenerative phenomena (Niki et al. 2005). In our study, 1,000 lM ClO4- cause the significant increase of MDA content (Fig. 3) suggest an increased lipid peroxidation in mitochondria, which might be associated damage to membrane may therefore trigger/propagate various pathological changes. Moreover, our study revealed that ClO4- enhanced the production of ROS in isolated mitochondria (Fig. 4), which provided strong evidence for the involvement of oxidative stress in ClO4--
induced toxicity. Considering the results mentioned above, we suppose that under the oxidative stress of ClO4-, the excess of ROS generated which leading to loss of Dwm, and accounting for the mPTP opening that may cause a disturbance of mitochondrial function (Jezek and Hlavata 2005; Valko et al. 2006). In addition, excessive ROS accumulation may result in the enhancement of DNA damage (Liu et al. 2010; Shao et al. 2012). Here, when incubated with ClO4-, DNA fragmentation was observed in isolated mitochondria (Fig. 5). These findings suggested that in addition to the loss of Dwm, the opening of mPTP and the promotion of polarization of the inner membrane, the generation of ROS are linked to DNA fragmentation in isolated mitochondria when incubated with ClO4-. On the basis of these findings, it is possible to hypothesize that ClO4- cause injury (even apoptosis or necrotize) on primary cultures hepatocytes of Carassius auratus through an oxidative stress-trigger pathway with mitochondria. In eukaryotes, mitochondrial respiratory chain is a significant source of ROS and impairment of the respiratory chain complexes is known to increase the ROS production (Indo et al. 2007; Houtkooper and Vaz 2008; Mustafa et al. 2011). However, mitochondria are not only sources but also vulnerable targets of ROS. It has, therefore, been proposed that oxidative to phosphorylation components may stimulate further ROS production, a vicious cycle that can eventually lead to mitochondrial collapse and cell death (Paranagama et al. 2010). Mitochondrial complex I, is the important source of ROS production, which inactivation may cause mitochondrial dysfunction. In the present
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study, the ClO4--mediated inhibitory activity of respiration exerted by exposure of cultured mitochondria was largely correlated to the specific inhibition of complexes I and IV (Fig. 6), did not result in a comparable degree of inhibition of the global respiration chain, which suggest that the inhibitory binding site of ClO4- likely locates only in the membranous part of the complex I and IV. Meanwhile, it is the ClO4--mediated inhibition of the respiratory chain complexes I that leading to enhanced ROS production and unbalance of the intracellular redox state may account for the cytotoxicity of ClO4- (Fig. 7). The mechanism of complex IV inhibition induced by ClO4- is not completely elucidated. Since many of theses processes are inter-dependent (Sen et al. 2007). Here it is presumable that during ClO4- damage to mitochondria involving mitochondrial phospholipid, is the primary event that leads to dissipation of H? ion gradient with consequent loss of mitochondrial transmembrane potential, and complex IV inhibition was unrelated to oxidative damage, or more probably, was irreversible (Fig. 7). Furthermore, from the available data of this study, it is also not possible to conclude whether increased ROS production by isolated mitochondria related to the cause or the effect of complex IV inactivation. But some possible mechanisms must be raised, such as failed transcription and translation of key subunits of the enzyme or reduced substrate concentration. Here, we demonstrated that superoxide mitochondrial production and complex I and IV activity seemed to be the earliest alternations in isolated liver mitochondria when incubated with ClO4-. For both complex I and IV are involved in the oxidoreduction chemistry, possess an intrinsic catalytic activity in the intramolecular electron transfer (Ohnishi et al. 2008). Generally, the respiratory chain of isolated mitochondria respiring under uninhibited conditions produce significant amount of ROS only during succinate oxidation by complex II, but the site of ROS production is not intrinsic to complex II (Fig. 7). In this situation, ROS are produced from complex I during the reversed flow of electrons derived from succinate oxidation to reduce NAD (Paranagama et al. 2010). As a result, we suppose that ClO4- exert its effect by perturbing the oxidoreduction chemistry of the quinone binding site causing leakage of electrons to O2 with formation of the superoxide anion radical largely from complexes I (Dro¨se et al. 2008). Indeed, ROS are generated by ClO4--dependent perturbation of the respiratory chain complexes I, but are also causally linked to their inhibition (Cela et al. 2010).
Conclusions In summary, this work successfully evaluates the effect of ClO4- on isolated mitochondria to understand more
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completely the mechanisms of ClO4- toxicity. The experimental results suggest that ClO4- exhibit multiple actions: (a) ClO4- induced liver mitochondrial oxidative stress, and subsequently caused a gradual opening of PTP leading to mitochondrial swelling and lipid peroxidative membrane damage; (b) ClO4- has a conspicuous inhibition of electron transport chain (ETC) activity and the damage was largely correlated to the specific inhibition of complexes I and IV. The present studies suggest ClO4-induced mitochondrial alternation is related to oxidative stress and damage of membrane. However, the contribution of these processes to mitochondrial dysfunction and degeneration associated with ClO4- needs to be explored further in appropriate models. To the best of our knowledge, there are few reports on the study of the cytotoxicity of ClO4- on mitochondria in vitro. Hence, the specific reasons and mechanisms for the conclusion above are not clear yet. Further studies are required to elucidate the precise mechanism of perchlorateinduced oxidative stress in isolated liver mitochondria. Acknowledgments The authors gratefully acknowledge the financial supports from the National Hi-Tech. Research and Development, program (863) of China (No. 2007AA06Z418), the National Natural Science Foundation of China (Nos. 20577036, 20777058, 20977070).
Conflict of interest of interest.
The authors declare that they have no conflict
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Perchlorate-induced oxidative stress in isolated liver mitochondria Xiaohu Zhao • Peijiang Zhou • Xiu Chen Xi Li • Ling Ding
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Accepted: 9 August 2014 / Published online: 20 August 2014 Ó Springer Science+Business Media New York 2014
Abstract As a new threat to environment all through the world, perchlorate (ClO4-) was predominantly a thyrotoxin, and its toxic manifestations in non-thyroid were also documented. To date, little is known about the effect of ClO4- on cell and organelle. To reveal the toxicity of ClO4- on living organism in-depth, mitochondria isolated from liver of Carassius auratus were incubated with different concentrations of ClO4-. The results demonstrated that ClO4--induced mitochondrial oxidative stress, and subsequently caused a gradual opening of permeability transition pore leading to mitochondrial swelling and lipid peroxidative membrane damage. ClO4- has a conspicuous inhibition of electron transport chain activity which largely correlated to complexes I and IV. The investigations clearly demonstrated the oxidative stress of ClO4- in mitochondria, may well reveal cytotoxic effects in vitro that merit further investigation.
X. Zhao P. Zhou (&) X. Chen X. Li L. Ding Hubei Biomass-Resource Chemistry and Environmental Biotechnology Key Laboratory, College of Resources and Environmental Sciences, Wuhan University, Wuhan 430072, Hubei, China e-mail: [email protected]; [email protected] X. Zhao e-mail: [email protected] X. Chen e-mail: [email protected] X. Li e-mail: [email protected] L. Ding e-mail: [email protected] X. Zhao Micro-element Research Center, Huazhong Agricultural University, Wuhan 430070, China
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Keywords Perchlorate Oxidative stress Liver Mitochondria
Introduction Perchlorate (ClO4-) and its salts are powerful oxidant that are widely used in rocket fuels, propellants, explosives (Attanasio et al. 2011). Because of the relatively high solubility in water, ClO4- would be produced once perchlorate salts released into aqueous systems. Due to the rapid mobility, stability, and nonreactivity, ClO4- in environment can persist for years to decades. Studies indicated that perchlorate is also found in a variety of foods and food crops (Sanchez et al. 2006; Murray et al. 2008), the milk from cows (Sanchez et al. 2008), and human breast milk (Pearce et al. 2007). Perchlorate is regarded as a new emerging persistent inorganic contaminant (Can˜as et al. 2006), and a new threat to environment all through the world. Due to potential toxicity, perchlorate has become serious threat to human health. To determine the toxic effects of perchlorate, experiments in vivo and pathological studies were used frequently. Studies have reported that perchlorate in the human body interferes with the uptake of iodine into the thyroid and may interfere with the development of the skeletal system and the central nervous system of infants (Borjan et al. 2011). In addition, treatment with high concentration of ClO4- significantly influenced the growth of preantral follicles and antral follicles, as well as other organisms (Bradford et al. 2005). However, based on its toxicity on living organism, there are relatively few studies on toxic effects of ClO4- in vitro, or has not been investigated in-depth. Moreover, little is known about the mechanism by which ClO4- act on cellular level or molecular level.
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Due to ClO4- in aquatic systems can persist for many years, fish in contaminated water may be affected by ClO4-. The liver is known to be the most important target organ involved in metabolism and detoxification for xenobiotics in vertebrates. The mitochondria is an important organelle which, in addition to ATP synthesis, participate in cytosolic calcium homeostasis regulation and cell signaling (Zhang et al. 2008). It also participates in important regulative processes like apoptosis. Hence, the maintenance of mitochondrial activity is the key aspect of cellualr survival particularly under stressed conditions (Parihar et al. 2009). The freshwater fish Carassius auratus, commonly found in China and widely used in aquatic toxicology, was chosen as the test organism (Feng et al. 2013). Previous research in our laboratory revealed that ClO4influenced the metabolic activity of mitochondria and cell (Zhao et al. 2011), and ClO4- increased ROS and MDA in hepatocytes isolated from Carassius auratus. To investigate mitochondrial functional alternation related to ClO4-induced oxidative stress, mitochondria isolated from liver of Carassius auratus were incubated with different concentrations of ClO4-. The present work is the first attempt to explore the influence of ClO4- stress on oxidative stress associated mitochondria, which would contribute to a better understanding of the toxic effect of ClO4- to aquatic organisms.
Materials and methods Chemicals Analytical-reagent grade NaClO4 was obtained from Tianjin Kermel Chemical Reagent Co., Ltd (Tianjin, People’s Republic of China). The mitochondria isolation medium A contained 250 mM sucrose, 50 mM Tris–HCl, 1 mM Na2EDTA, 0.1 % BSA, 1 mM 2-mercaptoethanol, 10 mM Tris–HCl, pH7.5 and isolation medium B contained 250 mM sucrose, 50 mM Tris–HCl, pH 7.5. The lipid peroxidation (MDA) detection kit was purchased from Nanjing jiancheng Bioengineering Institute (Nanjing, China). All other reagents were of the purest grade available and purchased from Sigma unless indicated otherwise. Fish sampling The present study was conducted in accordance with the Guiding Principles in the Use of Animals in Toxicology. Carassius auratus (0.2–0.3 kg each) were provided by Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China). They were kept in 300 L fiber-glass tanks with aerated, dechlorinated water (20 ± 2 °C, 12 h
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light:12 h dark cycles) for about 10 days before hepatocytes isolation. Fish was sacrificed by severing their spinal cord and the liver was removed immediately for the isolation of mitochondria. Isolation of mitochondria The mitochondria were isolated according to the methods of Liu et al. (2000) and Zhu et al. (2006), with a slight modify. First, removed liver tissue from Carassius auratus, weighed and rinsed with cold sterilized isolation mediun A, and then minced, homogenized, and centrifuged at 1,000 rpm for 15 min. The clear supernatant was centrifuged again for another 15 min at 1,000 rpm. Next, the pellet was suspended in isolation medium A and centrifuged at 15,000 rpm for 10 min in a new tube. The resulting pellet was washed twice with isolation medium B, centrifuged for 30 min at 15,000 rpm. The purified mitochondria were suspended again in isolation medium B. All of the above operations were performed aseptically at 0–4 °C. We maintained the equal concentration of mitochondria protein at 1 mg protein mL-1 by adding the suspension medium. Protein content was measured by the method described in ‘‘Protein assay’’ section. Mitochondrial swelling assays Mitochondrial osmotic volume changes due to colloidal effects of solute flux into and out of the mitochondrial matrix was measured by monitoring the absorbance at 540 nm (Zhao et al. 2010). The assays were performed in 1 mL of incubation buffer (200 mM sucrose, 5 mM succinate, 2 lM rotenone, 10 lM EGTA, 3 mM HEPES, 1 mM KH2PO4, pH 7.4) with isolated mitochondria (1 mg protein), which were added into a 96-well microplate and incubated with ClO4- (0, 0.1, 1, 10, 100, 1,000 lM) at 30 °C. Absorbance at 540 nm was monitored using a microplate spectrophotometer (Sunrise, Tecan, Switzerland) at 2 min time intervals for 30 min. Here, the highest and non-effective doses of ClO4- were according to our previous study. Measurement of fluorescence polarization in isolated Mitochondria Fluorescence polarization was measured using 1,6-diphenyl-1,3,5-hexatriene (DPH) (Li et al. 2009). Briefly, after mitochondrial fractions (1 mg) were incubated with concentrations of ClO4- as mentioned above. Then washed and incubated in 1 mL 1 9 10-6 M DPH solution at pH 7.4, at 25 °C for 30 min. Fluorescence polarization was measured by a spectrofluorometer equipped with a polarization attachment at excitation 362 nm, and emission
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432 nm at 25 °C. The degree of fluorescence polarization (P) was calculated as: P ¼ ðIvv GIvh Þ=ðIvv þ GIvh Þ where Ivv and Ivh are the fluorescence intensities of emitted light with vertical excitation and vertical (horizontal) emission polarizer, respectively. The emission grating factor (G) corrected for the parallel diffraction anomalies introduced by the glass of the phototube was calculated as: G ¼ Ihv =Ihh Ihv (hv) is the intensity with horizontal excitation with horizontal (vertical) emission polarizer. Mitochondrial membrane fluidity (1/g) was calculated as: 1=g ¼ ð0:46 PÞ=2P Mitochondrial MDA assays Malondialdehyde (MDA) concentration was detected in isolated liver mitochondria using thiobarbituric acid reactive substances (TBARS) according to the protocol of MDA kit. In brief, 1 mL mitochondrial suspensions were incubated with concentrations of ClO4- as mentioned above, for 30 min at 25 °C. Then the supernatant fractions were collected and lipid peroxidation was estimated spectrophotometrically at 530 nm. The MDA concentration was expressed as nmol TBARS mg-1 protein. Measurement of ROS production in isolated mitochondria ROS production in isolated mitochondria was measured using a dichlorofluorescin diacetate (DCFH2-DA) fluorescence probe. Briefly, mitochondrial fractions (1 mg) were incubated with ClO4- (as mentioned above) for 30 min at 25 °C. After washing, mitochondrial suspensions were incubated with 20 lM of DCFH2-DA for 20 min at 30 °C. At the end of the incubation, mitochondria were washed with fresh buffer at 12 000 rpm for 15 min at 4 °C and resuspended. After, the fluorescence intensity was measured immediately using a microplate reader (Synergy HT, Bio-Tek Instruments, USA). Excitation and emission wavelengths were 490 and 530 nm, respectively (Zhang et al. 2008; Zhao et al. 2010). The ROS concentration was expressed as DCF fluorescence intensify.
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buffer (10 mM Tris–HCl, pH 7.4, 10 mM EDTA, 100 mM NaCl, 200 lg mL-1 proteinase K and 0.5 % SDS). Then, the lysate was extracted with phenol: chloroform: isoamyl alcohol (25:24:1) and centrifuged at 10 000 rpm for 5 min at 4 °C. Next, DNA was precipitated with 3 M sodium acetate and 100 % cold ethanol and deposited for 12 h at -20 °C. Then the precipitated was centrifuged at 12,000 rpm for 15 min at 4 °C. The DNA pellets were washed with 70 % ethanol, air-dried, and dissolved in Tris– EDTA buffer (10 mM Tris, 1 mM EDTA, pH 7.4). After that, 5 mg L-1 RNase A was added to each sample to remove RNA. Samples were electrophoresed on a 1.2 % agarose gel (containing 10 lg mL-1 ethidium bromide) for 1.2 h at 60 V. After eletrophoresis, the DNA ladder was visualized by Bioimaging System (Dong et al. 2009). Measurement of the mitochondrial respiratory chain complexes activity After incubated with various of ClO4- as mentioned above, washed, and mitochondrial fractions (1 mg) were freezed and thawed three times to fully expose the enzymes to substrates and achieve maximal activities. Complex I activity was measured in the presence of decylubiquinone and succinate as the rotenone sensitive decrease in NADH at 340 nm. Complex II activity was measured by the rate of 2,6-DCPIP reduction at 600 nm (Albayrak et al. 2003; Lemarie et al. 2009). Complex III activity was measured by following the initial antymicin A-sensitive rate of cytochrome c reduction at 550 nm in the presence of rotenone and decylubiquinone (Cela et al. 2010). Complex IV activity was assayed by following the decrease in absorbance due to the oxidation of previously reduced cytochrome c at 550 nm according to the method described by Rustin (1994). The activities of mitochondrial respiratory chain complexes were expressed as nmol/ min/mg protein. Protein assay Protein content was assayed according to Bradford (1976), using bovine serum albumin (Hyclone, USA) as a standard. Statistical analysis
DNA fragmentation assay The determination of DNA fragmentation pattern (DNA ladder) was carried out by agarose gel electrophoresis. After incubation as mentioned above, mitochondria were washed and then lysed for 1 h at 25 °C with 1 mL lysis
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Data presented are mean ± standard deviation (S.D.) of three different experiments. The statistical analysis was performed using one-way analysis of variance (ANOVA SPSS 18.0, Chicago, IL, USA) followed by Dunnett-t-test. The significance level was ascertained at P \ 0.05.
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Results -
ClO4 induced isolated mitochondrial swelling The swelling of mitochondria reflected the opening of mitochondrial ion channels and membrane pores. As shown in Fig. 1, when in absence of ClO4-, mitochondria swells very slowly. However, the addition of ClO4- caused a concentration -dependent manner in mitochondrial swelling. A rapid swelling was not observed until the presence of 100–1,000 lM ClO4-. Effects of ClO4- on fluidity of isolated mitochondrial membrane The effect of increasing concentrations of ClO4- on DPH polarization of mitochondrial membrane was shown in
Fig. 2. No significant changes occurred in DPH polarization until 1,000 lM ClO4- treated. The result revealed that ClO4- can decrease the fluidity of mitochondrial membrane, but only in high concentration-treated.
Assessment of MDA in isolated mitochondria Levels of MDA, the last product of lipid breakdown caused by oxidative stress, were assessed in mitochondria from the control group. As shown in Fig. 3, MDA levels were significantly increased in the mitochondria treated with 1,000 lM ClO4- group, compared to the normal control. However, no significant difference was recorded between low-dose groups and the normal control group.
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Generation of ROS of isolated mitochondria Treatment of mitochondria with increasing concentrations of ClO4- resulted in a significant enhancement of the DCFrelated fluorescence indicating alterations of the intracellular redox state. As shown in Fig. 4, the DCF-fluorescence signal no or faintly changed after incubated with 0.1–100 lM ClO4-. However, the same result as polarization of mitochondrial membrane, when compared to the control group, the production of ROS was elevated only when treated with 1,000 lM ClO4-. Effects of ClO4- exposure on DNA fragmentation in isolated mitochondria The DNA of apoptotic cells presented as a regular ladder, which is typical of cells undergoing apoptosis. DNA fragmentation in isolated mitochondria was observed as shown in Fig. 5. Compare with the control, negligible DNA fragmentation produced when treated with 0.1–10 lM ClO4-. However, when mitochondria exposed to 100–1,000 lM ClO4-, DNA in mitochondria was cut into fragment. Effects of ClO4- exposure on the activities of mitochondrial complex To locate the inhibition sites in the mitochondrial respiratory chain, the effect of ClO4- was tested on the specific activities of complexes. The data in Fig. 6 show that there was a statistically decline of complex I and IV activity in
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Fig. 6 Effects of perchlorate on mitochondrial respiratory complex I, II, III and IV activities. Data are reported as mean ± SD, N = 3. Asterisks indicate significant difference from control (*P \ 0.05; **P \ 0.01)
isolated mitochondria when exposured to high concentrations of ClO4-, compared with the control. The activity of complex I reduced approximately 22.4 and 21.8 % when incubated with 100, 1,000 lM ClO4-, respectively. And the activity of complex IV reduced approximately 30.0, 37.7 and 48.4 % when incubated with 10, 100, 1,000 lM ClO4-, respectively. Interestingly, by an unknown mechanism, both the activity of complex II and complex III were unaffected by ClO4- at all the incubation-concentration tested.
Discussion Recently, the effects of ClO4- have been studied on diverse biological systems. Experiments in vivo showed that treatment with high concentration of ClO4- significantly influenced the growth of preantral follicles and antral follicles (Baldridge et al. 2004), as well as other organisms (Bradford et al. 2005). Moreover, growth indices of some organs/tissues were diminished by high concentration, but enhanced at low (Park et al. 2006). In previous work, we found that ClO4- influenced the metabolic activity of mitochondria and cell (Zhao et al. 2011). In addition, in the presence of high concentration of ClO4-, the primary cultured hepatocytes isolated from Carassius auratus started to shrink and became irregular in shape, displayed nuclear fragmentation, membrane blebbing, endoplasmic reticulum dilation, mitochondrial swelling or vacuolization, and the number of apoptotic cells and necrotic cells were significantly increased. Liver, which are particularly rich in mitochondria and that rely on aerobic ATP production are expected to be
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Fig. 7 Schematic view of the proposed mechanisms of ClO4--mediated ‘‘mitotoxicity’’. The action of ClO4- leading to collapse of MMP, consequent decrease of the ATP synthesis and the production of ROS are indicated in the model
more vulnerable to the action of toxin. Therefore, in the present study, to investigate mitochondrial functional alternation related to ClO4--induced oxidative stress and the cytotoxicity, mitochondria isolated from liver of Carassius auratus were incubated with different concentrations of ClO4- in this study. Mitochondria are involved in a large variety of cellular functions which are largely determined by the inner membrane potential. The change of the inner membrane potential may induce a series of reaction which cause the apoptosis. In addition, depolarization of mitochondria is an early sign of mitochondrial dysfunction and often recedes many other signs of cell injury (Muramatsu et al. 2007). In this study, the promotion of polarization of the inner membrane (Fig. 2), and mitochondrial swelling (Fig. 1) were observed, indicating change in membrane permeability. It is concluded that ClO4--induced mitochondrial permeability transition pore (mPTP) opening, which may caused destruction to hepatocytes. MDA is a terminal product of polyunsaturated fatty acid peroxidation, often used as a marker of oxidative damage associated with degenerative phenomena (Niki et al. 2005). In our study, 1,000 lM ClO4- cause the significant increase of MDA content (Fig. 3) suggest an increased lipid peroxidation in mitochondria, which might be associated damage to membrane may therefore trigger/propagate various pathological changes. Moreover, our study revealed that ClO4- enhanced the production of ROS in isolated mitochondria (Fig. 4), which provided strong evidence for the involvement of oxidative stress in ClO4--
induced toxicity. Considering the results mentioned above, we suppose that under the oxidative stress of ClO4-, the excess of ROS generated which leading to loss of Dwm, and accounting for the mPTP opening that may cause a disturbance of mitochondrial function (Jezek and Hlavata 2005; Valko et al. 2006). In addition, excessive ROS accumulation may result in the enhancement of DNA damage (Liu et al. 2010; Shao et al. 2012). Here, when incubated with ClO4-, DNA fragmentation was observed in isolated mitochondria (Fig. 5). These findings suggested that in addition to the loss of Dwm, the opening of mPTP and the promotion of polarization of the inner membrane, the generation of ROS are linked to DNA fragmentation in isolated mitochondria when incubated with ClO4-. On the basis of these findings, it is possible to hypothesize that ClO4- cause injury (even apoptosis or necrotize) on primary cultures hepatocytes of Carassius auratus through an oxidative stress-trigger pathway with mitochondria. In eukaryotes, mitochondrial respiratory chain is a significant source of ROS and impairment of the respiratory chain complexes is known to increase the ROS production (Indo et al. 2007; Houtkooper and Vaz 2008; Mustafa et al. 2011). However, mitochondria are not only sources but also vulnerable targets of ROS. It has, therefore, been proposed that oxidative to phosphorylation components may stimulate further ROS production, a vicious cycle that can eventually lead to mitochondrial collapse and cell death (Paranagama et al. 2010). Mitochondrial complex I, is the important source of ROS production, which inactivation may cause mitochondrial dysfunction. In the present
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study, the ClO4--mediated inhibitory activity of respiration exerted by exposure of cultured mitochondria was largely correlated to the specific inhibition of complexes I and IV (Fig. 6), did not result in a comparable degree of inhibition of the global respiration chain, which suggest that the inhibitory binding site of ClO4- likely locates only in the membranous part of the complex I and IV. Meanwhile, it is the ClO4--mediated inhibition of the respiratory chain complexes I that leading to enhanced ROS production and unbalance of the intracellular redox state may account for the cytotoxicity of ClO4- (Fig. 7). The mechanism of complex IV inhibition induced by ClO4- is not completely elucidated. Since many of theses processes are inter-dependent (Sen et al. 2007). Here it is presumable that during ClO4- damage to mitochondria involving mitochondrial phospholipid, is the primary event that leads to dissipation of H? ion gradient with consequent loss of mitochondrial transmembrane potential, and complex IV inhibition was unrelated to oxidative damage, or more probably, was irreversible (Fig. 7). Furthermore, from the available data of this study, it is also not possible to conclude whether increased ROS production by isolated mitochondria related to the cause or the effect of complex IV inactivation. But some possible mechanisms must be raised, such as failed transcription and translation of key subunits of the enzyme or reduced substrate concentration. Here, we demonstrated that superoxide mitochondrial production and complex I and IV activity seemed to be the earliest alternations in isolated liver mitochondria when incubated with ClO4-. For both complex I and IV are involved in the oxidoreduction chemistry, possess an intrinsic catalytic activity in the intramolecular electron transfer (Ohnishi et al. 2008). Generally, the respiratory chain of isolated mitochondria respiring under uninhibited conditions produce significant amount of ROS only during succinate oxidation by complex II, but the site of ROS production is not intrinsic to complex II (Fig. 7). In this situation, ROS are produced from complex I during the reversed flow of electrons derived from succinate oxidation to reduce NAD (Paranagama et al. 2010). As a result, we suppose that ClO4- exert its effect by perturbing the oxidoreduction chemistry of the quinone binding site causing leakage of electrons to O2 with formation of the superoxide anion radical largely from complexes I (Dro¨se et al. 2008). Indeed, ROS are generated by ClO4--dependent perturbation of the respiratory chain complexes I, but are also causally linked to their inhibition (Cela et al. 2010).
Conclusions In summary, this work successfully evaluates the effect of ClO4- on isolated mitochondria to understand more
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completely the mechanisms of ClO4- toxicity. The experimental results suggest that ClO4- exhibit multiple actions: (a) ClO4- induced liver mitochondrial oxidative stress, and subsequently caused a gradual opening of PTP leading to mitochondrial swelling and lipid peroxidative membrane damage; (b) ClO4- has a conspicuous inhibition of electron transport chain (ETC) activity and the damage was largely correlated to the specific inhibition of complexes I and IV. The present studies suggest ClO4-induced mitochondrial alternation is related to oxidative stress and damage of membrane. However, the contribution of these processes to mitochondrial dysfunction and degeneration associated with ClO4- needs to be explored further in appropriate models. To the best of our knowledge, there are few reports on the study of the cytotoxicity of ClO4- on mitochondria in vitro. Hence, the specific reasons and mechanisms for the conclusion above are not clear yet. Further studies are required to elucidate the precise mechanism of perchlorateinduced oxidative stress in isolated liver mitochondria. Acknowledgments The authors gratefully acknowledge the financial supports from the National Hi-Tech. Research and Development, program (863) of China (No. 2007AA06Z418), the National Natural Science Foundation of China (Nos. 20577036, 20777058, 20977070).
Conflict of interest of interest.
The authors declare that they have no conflict
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