Oxidative phosphorylation pogil answer key pdf printable 2017 pdf

Oxidative phosphorylation pogil answer key pdf printable 2017 pdf

Oxidative phosphorylation pogil answer key pdf printable 2017 pdf

Pb2+ exposure in humans occurs mainly through air inhalation, food and water uptake which has been shown to be generally associated with numerous body functions such as the central and peripheral nervous systems, the red blood cells, the kidneys and the liver. It has been reported that the liver is the storage site and an important primary target in Pb2+ toxicity, and the hepatotoxicity of Pb2+ could be resulted from the impairment of the liver mitochondria. In this study, several mitochondrial dysfunctions following the addition of Pb2+ (10?160 M) were investigated. We found that Pb2+ inhibited the enzyme activities of mitochondrial respiratory complexes and complex III was the major source of Pb2+-induced significant reactive oxygen species (ROS) formation. As a consequence, our results showed that Pb2+ induced significant progress in mitochondrial lipid peroxidation, adenosine triphosphate (ATP) consumption and glutathione (GSH) oxidation. On the other hand, Pb2+ induced marked changes in mitochondrial permeability transition (MPT) accompanied by mitochondrial swelling, mitochondrial membrane potential collapse, mitochondrial membrane fluidity decrease and cytochrome c (Cyt c) release. Additionally, several mitochondrial MPT inhibitors and chelators were utilized to determine the possible interaction sites of Pb2+ on mitochondria. In general, our data supported that the Pb2+-induced liver toxicity was a result of the disruptive effect on the mitochondrial respiratory complexes. This disruptive effect caused oxidative stress and MPT, which led to mitochondrial dysfunctions and even cell death signalling via mitochondrial permeability transition pore (MPTP) opening and Cyt c release.Lead is one of the poisonous and ubiquitous heavy metals. Its multiple industrial, domestic, agricultural, medical and technological applications (lead-acid batteries, ammunitions, metal products and devices to shield X-rays) have led to its wide distribution in the environment. Emissions of lead into the environment occur via a wide range of processes and pathways, including through air (e.g. during combustion, extraction and processing), through surface waters (via runoff and releases from storage and transport) and through soil (and hence into ground waters and crops).1 Lead is known to interfere with a number of body functions such as the central and peripheral nervous systems, the red blood cells and their precursors, the kidneys and the liver. In the case of lead intoxication, the central nervous system is the most vulnerable target. Headache, poor attention span, irritability, loss of memory and dullness are the early symptoms of the effects of lead exposure on the central nervous system.2,3 Low levels of exposure have been linked with increased risks for numerous diseases such as reading problem, hearing loss, tooth decay, spontaneous abortions, and cardiovascular disease.4 Moreover, Pb2+ exposure also causes anaemia, hypertension, immunotoxicity, renal impairment and toxicity in the reproductive organs.5,6 The hepatocarcinogenic effects of Pb2+ reported in animal toxicology studies have led to new research into the biochemical and molecular aspects of Pb2+ toxicology. Mitochondria were the important intracellular target sites for Pb2+.7 Mitochondria are the major source of ROS in most mammalian cell types and are responsible for the production of 60?80% of H2O2 in cells under normal conditions which can be increased under pathological conditions and through the inhibition of mitochondrial electron transfer chain (ETC) complexes.7,8 Several studies have demonstrated that Pb2+-induced ROS production and oxidative stress which is postulated to be one of the possible toxicology mechanisms. Moreover, Pb2+ induces mitochondrial impairment indirectly by: (i) decreasing the expression of the mitochondrial calcium uniporter, destabilizing intracellular calcium homeostasis;9 (ii) increasing DNA damage;10 and (iii) activating redox sensitive kinases.11Our work aimed at evaluating the biological effects of Pb2+ on the mitochondrial oxidative stress and a series of mitochondrial permeability transition (MPT) related consequences like swelling, membrane potential collapse and membrane fluidity change. Furthermore, we utilized several MPT inhibitors to confirm the possible interaction sites of Pb2+ on mitochondria. We postulated that the cytotoxicity of Pb2+ was the result of its disruptive effect on the liver mitochondrial respiratory chain complex III and the subsequent formation of reactive oxygen species (ROS) and lipid peroxidation, which caused MPT pore (MPTP) opening and cytochrome c (Cyt c) release, the starting point of apoptosis signalling in liver cells.3-Morpholinopropanesulfonic acid (MOPS), 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB), adenosine diphosphate (ADP), rotenone, oligomycin, rhodamine 123 (Rh123), 1,6-diphenyl-1,3,5-hexatriene (DPH), cyclosporine A (CsA), ruthenium red (RR), ethylene glycol tetraacetic acid (EGTA), ethylene diamine tetraacetic acid (EDTA) and meso-2,3-dimercaptosuccinic acid (DMSA) were purchased from Sigma (St Louis, MO). Other reagents were of analytical reagent grade. All solutions were prepared with deionized water. Deionization water was prepared from a Milli-Q-RO4 water purification system (18.2 M cm?1, Millipore).Female Wistar rats (about 150 g) were purchased from the Hubei Center for Disease Control and Prevention (Wuhan, China), and housed in ventilated micro-isolator cages with free access to water and food in a constant temperature room (22 ? 2 ?C). Animals were handled according to the guidelines of the China Animal Welfare Legislation, as approved by the Committee on Ethics in the Care and Use of Laboratory Animals of the College of Life Sciences, Wuhan University.The isolation of liver mitochondria was done according to the literature with some modifications.12,13 Briefly, fresh rat liver samples were quickly removed and placed in a beaker, washed in ice-cold homogenization buffer: solution A (220 mM mannitol, 70 mM sucrose, 20 mM HEPES, 2 mM Tris-HCl and 0.1 mM EDTA, pH 7.4) twice. Then the liver tissue (about 2 g) was finely minced and suspended in 40 mL of solution A with 0.4% (w/v) bovine serum albumin (BSA) added. After chilling in ice-cold water, the suspension was homogenized with a glass handheld homogenizer and then centrifuged at 3000g for 4 min. The supernatant was carefully decanted and centrifuged at 17500g for 5 min. The pellet was diluted by gently resuspending in solution B (220 mM mannitol, 70 mM sucrose, 10 mM Tris-HCl and 0.1 mM EDTA, pH 7.4) and subjected to a further centrifugation at 17500g for 5 min twice. The final mitochondrial pellet was preserved in solution C (220 mM mannitol, 70 mM sucrose and 0.1 mM EDTA, pH 7.4). All of the above steps were strictly operated at 0?4 ?C and the isolated mitochondria were prepared within 3 h in order to guarantee high-quality fresh mitochondrial preparation. Mitochondrial protein concentrations were determined by the Biuret method. BSA was used as a standard for protein measurement.14,20 The mitochondrial ROS measurement was performed through flow cytometry by using DCFH-DA (2,7-dichlorodihydrofluorescein diacetate). Isolated liver mitochondria (0.5 mg protein per mL) were incubated with Pb2+ in respiration buffer MRB (2 mL, containing 0.32 mM sucrose, 10 mM Tris, 20 mM Mops, 50 mM EGTA, 0.5 mM MgCl2, 0.1 mM KH2PO4 and 5 mM sodium succinate, pH 7.4) for 15 min at 25 ?C. The samples with DCFH-DA (5 M) were incubated for 10 min and then determined through flow cytometry (BD, C6) equipped with a 488 nm argon ion laser and supplied with the Cytometer software. The signals were obtained using a 530 nm bandpass filter (FL-1 channel). Each determination is based on the mean fluorescence intensity of 50000 counts.15All assays were carried out in 200 L final volume with 4?5 g mitochondrial proteins. The absorbance was monitored by using a multimode microplate reader (Tecan Spark? 10M, Switzerland) for at least 3 plex I: Mitochondria were incubated with Pb2+ in 10 mM Tris-HCl buffer (pH 8.0) for 15 min at 25 ?C. After incubating with the reaction buffer containing 80 M 2,3dimethoxy-5-methyl-6-decyl-1,4-benzoquinone (DB), 1 mg mL?1 BSA, 3 mM NaN3, and 0.4 M antimycin for another 5 min, the oxidation of NADH (200 M) was monitored at 340 plex III: Mitochondria were incubated in 50 mM Tris-HCl buffer, pH 7.4, containing 1 mM EDTA, 250 mM sucrose, 3 mM NaN3, and 30 M oxidized Cyt c. Then 80 M ubiquinol was added and the reduction of Cyt c was measured at 550 nm.The samples including specific inhibitors, namely complex I (rotenone, 5 M) and complex III (antimycin, 4 M) were tested to determine the specificity of the respiratory complex activities.The activity of mitochondrial complex II (succinate dehydrogenase) was assayed through the measurement of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction.16 Briefly, 100 L of mitochondria (0.5 mg protein per mL) were suspended in MAB (200 mM sucrose, 5 mM succinate, 1 mM Tris, 1 mM Na3PO4?10H2O, 1 mM MOPS, 1 M EGTA, 2 M rotenone and 3 g mL?1 oligomycin, pH 7.4) and incubated with different concentrations of Pb2+ at 25 ?C for 15 min. MTT (0.4%) was added and incubated at 25 ?C for another 30 min. The product of purple formazan crystals was dissolved in 100 L DMSO, and the absorbance at 490 nm was measured with a microplate reader (VictorTM X5, PerkinElmer, America).The activity of mitochondrial complex IV (Cyt c oxidase) was evaluated using a Cyt c oxidase activity assay kit (GMS10014.2; Genmed Scientifics, Inc.).16 The colorimetric assay was based on the observation that a decrease in the absorbance of ferrocytochrome c at 550 nm was caused by its oxidation to ferricytochrome c by Cyt c oxidase. Isolated mitochondria (0.5 mg protein per mL) were suspended in MAB and incubated with Pb2+ at 25 ?C for 15 min. The suspension was then centrifuged at 17500g for 10 min. Briefly, 5 g of the mitochondrial protein (with or without Pb2+ incubated) were incubated with 100 mM reduced ferrocytochrome c. The Cyt c oxidase activity was measured by using a UV-Vis spectrophotometer (SQ-4802; UNICO, Dayton, NJ).The extent of mitochondrial membrane lipid peroxidation was assessed by the consumption of oxygen using a Clark-type electrode thermostated at 25 ?C. Mitochondria (1 mg protein per mL) were injected into the stirred lipid peroxidation medium (1 mL, containing 175 mM KCl, 10 mM Tris-HCl and 3 M rotenone, pH 7.4).17 Rotenone in the medium could suppress mitochondrial respiration induced by endogenous substrates during the experiment. The membrane lipid peroxidation was initiated by adding 1 mM ADP and 0.1 mM Fe2+. Iron(ii) solution must be prepared immediately before using and protected from light.The malondialdehyde (MDA) content of mitochondria was also measured to assess the lipid peroxidation by using a Lipid Peroxidation MDA Assay Kit (Beyotime, Hangzhou, China), according to the manufacturer's protocol.18 During oxidative stress, the reaction of MDA combined with TBA creates TBA-MDA adducts which are recorded spectrophotometrically at 550 nm at 25 ?C.The isolated mitochondrial ATP content was assessed by luciferin?luciferase reaction with a commercially available ATP Assay Kit (Beyotime, S0026). The luminescence intensity from the reaction was proportional to the ATP concentration in the assay solution and measured with a microplate reader at 25 ?C (VictorTM X5, PerkinElmer, America).The GSH content was determined using DTNB as the indicator by the spectrophotometric method in isolated mitochondria. Mitochondria (0.5 mg protein per mL) were incubated with Pb2+ for 15 min at 25 ?C and then added into 0.04% DTNB (dissolved in 0.1 mM phosphate buffer, pH 7.4) in a total volume of 3.0 mL. The developed yellow color was seen at 405 nm using a microplate reader (VictorTM X5, PerkinElmer, America). The GSH content was expressed as g per mg per protein.Mitochondrial swelling was assessed through the absorbance changes by using a UV-Vis spectrophotometer (UV-6100PC; MAPADA, Shanghai, China) set at 540 nm for 600 s at 25 ?C.19 Mitochondria (0.5 mg protein per mL) were suspended in 2.0 mL MAB and treated with different concentrations of Pb2+. The reduction of absorbance is related to the increase in mitochondrial swelling. In order to understand the interaction site or sites of high concentrations of Pb2+ on isolated mitochondria, the protective effects of CsA, ADP, DMSA, EDTA, EGTA and RR on Pb2+induced mitochondrial swelling were also detected.Isolated mitochondria (0.5 mg protein per mL) were suspended in MAB (0.2 mL) as described above. The fluorescence intensity of the fluorescent dye Rh123 was continuously visualized and monitored with a multilabel plate reader (VictorTM X5, PerkinElmer, America) set for 488 nm excitation and 535 nm emission filters at 25 ?C.20Fluidity changes in the mitochondrial membranes were evaluated by the changes in the fluorescence excitation anisotropy (r) of mitochondria-bound dyes.21,22 In our experiments, mitochondria (0.5 mg protein per mL) were suspended in MAB (2.0 mL) with probes DPH added and incubated for 30 min before measuring. The values of steady-state anisotropy could be obtained at ex = 362 nm, em = 454 nm with a LS55 fluorescence spectrometer (PerkinElmer, Norwalk, USA) at 25 ?C. The anisotropy (r) is defined by using the following equation:r = (I ? GI)/(I + 2GI)1where G = I/I is the correction factor for instrumental artifacts. The emission polarizer is oriented parallel () to the direction of the polarized excitation and the observed intensity is called I. Likewise, when the polarizer is perpendicular () to the excitation, the intensity is called I.Isolated mitochondria were suspended in respiration buffer MRB and incubated with different concentrations of Pb2+ at 25 ?C for 15 min. The suspension was then centrifuged at 17500g for 10 min. The supernatant was analyzed by using a Rat/Mouse Cyt c ELISA Kit according to the manufacturer's protocol.23 The 96-well plates were precoated with an anti-Cyt-c polyclonal antibody and 50 L samples were added and incubated at 25 ?C for 30 min. Then a HRP?antibody conjugate was added and incubated at 25 ?C for 30 min. A chromogenic agent was added to the wells and incubated at 25 ?C for 10 min sequentially. The wells were washed three times with washing buffer before each step mentioned above. Finally, 50 L of stop solution were added. The absorption at 450 nm was recorded by using a microplate reader (VictorTM X5, PerkinElmer, America).The rates of ROS production in liver mitochondria isolated from rats treated with Pb2+ are shown in Fig. 1. The rate of ROS production in the mitochondrial suspension was determined through the changes in the fluorescence intensity. Our study showed that the ROS level was increased in a concentration and time dependent manner. But a low concentration of Pb2+ (10 M) did not significantly increase ROS generation until 60 min (data not shown).ROS formation in Pb2+-treated mitochondria. ROS formation after the addition of various concentrations of Pb2+ (0, 20, 40 and 80 M) at intervals of (A) 10 min after addition, (B) 30 min after addition, (C) 60 min after addition. (D) Summary of ROS formation. Values represented as mean ? SD (n = 3). Statistical significance *P < 0.05; **P < 0.01; ***P < 0.001 of the deviation from the control group.For exploring the toxicity of Pb2+ on the respiratory chain, the assessments of the activities of mitochondrial respiratory chain enzyme complexes including complexes I, II, III and IV were performed gradually. As shown in Fig. 2, the addition of Pb2+ influenced the activities of mitochondrial respiratory chain enzyme complexes.Different concentrations of Pb2+ inhibited the enzyme activities of respiration chain complexes I (A), II (B), III (C), and IV (D). Values represented as mean ? SD (n = 3). Statistical significance *P < 0.05; **P < 0.01; ***P < 0.001 of the deviation from the control pared with the activity of complex I, exposure with a high concentration of Pb2+ could suppress mainly the activity of complex III (Fig. 2A and C). The succinate dehydrogenase activity (complex II) was assessed by MTT reduction after a 15 min incubation of mitochondria with different concentrations of Pb2+ (10, 20, 40 and 80 M). Fig. 2B showed a decrease in the mitochondrial reduction of MTT to formazan. As the terminal enzyme complex of the electron transport chain, complex IV (Cyt c oxidase) is where over 90% of oxygen was consumed, which largely controls the mitochondrial respiration.24 Our results showed no significant change in the activity of the complex IV enzyme during the incubation time (Fig. 2D).The membrane lipid peroxidation induced by the pro-oxidant pair ADP/Fe2+ was characterized by a two-phase kinetics in oxygen consumption: an initial lag phase and the rapid oxygen consumption phase. The lag phase was probably related to the time required for the generation of an adequate ion complex which is suggested to be responsible for the initiation of lipid peroxidation.25 Due to the oxidation of the polyunsaturated fatty acid acyl chain on membrane phospholipids by ROS, the oxygen content was rapidly decreased.26 As shown in Fig. 3A, the addition of Pb2+ induced an increase in the oxygen consumption rate drastically. Lipid peroxidation was also assayed by measuring the content of mitochondrial MDA. As shown in Fig. 3B, the amount of MDA significantly increased at high concentrations of Pb2+ (40 and 80 M).Effect of different concentrations of Pb2+ on the mitochondrial lipid peroxidation. c (Pb2+)/M (a?f): 0, 10, 20, 40, 80, and 160. (A) Pb2+ exacerbated the consumption of O2. (B) Effect of Pb2+ on the mitochondrial MDA content. Values represented as mean ? SD (n = 3). Statistical significance *P < 0.05; **P < 0.01 of the deviation from the control group.The most important function of mitochondria is the generation of ATP through oxidative phosphorylation.27 The determination of ATP levels can provide an indirect measure of the mitochondrial function. As shown in Fig. 4A, mitochondrial ATP levels were significantly decreased by Pb2+ in a concentration-dependent manner.GSH is an important antioxidant in mitochondria, which has a critical role in defense against the ROS and the maintenance of the sulfhydryl groups in the reduced state.28 Compared to the control group, the GSH levels of Pb2+-incubated mitochondria were decreased (Fig. 4B).As a sign of mitochondrial dysfunction, swelling is one of the most important indicators of the MPTP opening.29 As shown in Fig. 5A, compared to the control group, Pb2+ could enhance the permeabilization of the mitochondrial membrane to sucrose, thus leading to the occurrence of swelling with a dose- and time-dependent effect.(A) Swelling of succinate-energized rat liver mitochondria at different concentrations of Pb2+. c (Pb2+)/M (a?f): 0, 10, 20, 40, 80, and 160. (B) Effect of Pb2+ on MMP collapse. The intensity of Rh123 in MAB without mitochondria is shown as trace a. c (Pb2+)/M (b?g): 0, 10, 20, 40, 80, and 160. (C) Pb2+ decreased the fluidity of the mitochondrial inner membrane. c (Pb2+)/M (a?e): 0, 40, 80, 160, and 320. (D) Protective effect of CsA, ADP, EGTA, EDTA, DMSA and RR on mitochondrial swelling caused by 80 M Pb2+. Control (a), 80 M Pb2+ (b), 80 M Pb2+ and 1 M CsA (c), 80 M Pb2+ and 200 M ADP (d), 80 M Pb2+ and 100 M EGTA (e), 80 M Pb2+ and 500 M EDTA (f), 80 M Pb2+ and 100 M DMSA (g), 80 M Pb2+ and 1 M RR (h).The MMP in hepatocytes was measured with Rh123, a cationic lipophilic fluorescent dye, which can selectively permeate the inner membrane and retain in the polarized mitochondrial matrix, thus, trapping inside the sub-cellular organelle led to fluorescence quenching. When the MMP decreased, Rh123 was released into the medium and its fluorescence intensity would increase subsequently.30 As shown in Fig. 5B, the MMP significantly decreased with different concentrations of Pb2+.The MPTP opening was always accompanied by the changes in membrane fluidity, and this could reflect the dynamic properties of the mitochondrial membranes.31 We chose the probe DPH which may incorporate underneath the polar region of the phospholipid bilayer and in the partition between the two acyl chain layers of the membrane core. The DPH-binding sites were involved in the MPTP structure or regulation.32 Here, the increase of DPH anisotropy corresponded to the decrease of membrane fluidity (Fig. 5C). At the tested concentration range, the increase of Pb2+ induced the decrease of membrane fluidity at a higher level.As illustrated in Fig. 5D, the effects of CsA, ADP, EDTA, EGTA, DMSA and RR on the swelling of succinate-energized mitochondria were respectively studied to elucidate the toxic mechanism of Pb2+. CsA mainly interacted with the matrix space protein cyclophilin D (CyP-D), which was considered as a well-established inhibitor of MPTP opening.33,34 CsA (1 M) could strikingly prevent the swelling induced by Pb2+. ADP could modulate MPTP opening by controlling adenine nucleotide translocase (ANT) conformation.35 ADP (100 M) partially prevented the swelling induced by Pb2+. Furthermore, EDTA and EGTA, chelators for metal ions, could lead to the recovery of mitochondrial energy linked functions.36Both EGTA (25 M) and EDTA (100 M) had protective effects on the Pb2+-induced mitochondrial swelling. RR was a noncompetitive inhibitor of the mitochondrial Ca2+ uniporter which can abolish Ca2+ influx effectively.37 Pretreatment with 1 M RR could suppress mitochondrial swelling caused by 80 M of Pb2+. DMSA was approved in the 1960s by the FDA specifically for the removal or chelation of heavy metals. It is particularly effective at removing lead and mercury.38 Besides this, DMSA (100 M) could partially inhibit Pb2+-induced mitochondrial swelling.Cyt c is believed to reside solely in the mitochondrial intermembrane cristae spaces under normal physiological conditions. The opening of MPTP caused mitochondrial swelling and released the apoptosis-initiating factor and Cyt c from the intermembrane space to the cytosol, which activates the caspase family of proteases, the primary trigger leading to the onset of apoptosis.39,40 Table 1 showed that the release of Cyt c from isolated mitochondria incubated with Pb2+ was increased during the incubation period.Effect of Pb2+ on the Cyt c release of liver mitochondriaPb2+ (M)OD (450 nm)Cyt c (nM)00.15516 ? 2200.20818 ? 3400.22621 ? 3800.26724 ? 4*1600.27525 ? 5**As subcellular organelles, isolated mitochondria samples offer a number of advantages over intact cells, particularly by their ability to manipulate the access to substrates and alter the local environment.41,42 Comprehensive research of mitochondria was helpful to understand the cellular and molecular mechanisms involved in the toxicity of xenobiotics. Through oxidative phosphorylation, mitochondria play their essential role to supply the cell with metabolic energy in the form of ATP.43 Following the addition of Pb2+ into isolated liver mitochondria, a rapid increase in ROS (H2O2) formation occurred (Fig. 1), which suggested the probable role of mitochondrial H2O2 in hepatotoxicity associated with Pb2+. Mitochondria are also the primary source of cellular ROS, especially generated in the respiratory chain complexes I and III, where electrons derived from NADH and ubiquinone can directly react with oxygen or other electron acceptors and generate free radicals.44 Therefore, one of the aims of this study was to identify the site of mitochondrial ROS production.To distinguish the involvement of complex I and/or III, we measured the rate of both the complex I substrate and complex III substrate supported mitochondrial hydrogen peroxide production in liver mitochondria under the treatment of different concentrations of Pb2+ (Fig. 2A and C). The high level of Pb2+ may impair complex III resulting in the increase of the leakage of electrons and generating more ROS, which ultimately leads to mitochondrial dysfunction. Our result showed that Pb2+ significantly reduced the function of complex II (Fig. 2B) and probably the inhibition of this enzyme contributes to Pb2+ toxicity. On the other hand, Pb2+ had little impact on the activity of complex IV(Fig. 2D).Lipid peroxidation is generally thought to be the major mechanism of biomembrane injury promoted by ROS. Since lipids within the mitochondrial membrane contain components of the electron transport chain, the lipid peroxidation partially reveals its influences on the electron transport chain.45 We found that Pb2+ indeed exerted a promotion effect on oxygen consumption and increased the content of MDA, suggesting the induction of lipid peroxidation in the isolated mitochondria (Fig. 3). Thus, the partial mechanism was proposed that the Pb2+-mediated inhibition of respiratory chain complexes was linked to the enhanced ROS production, the latter resulting in lipid peroxidation.Our investigation showed that after the addition of different concentrations of Pb2+, the rate of ATP production decreased down to 65% (Fig. 4A). The reduction of mitochondrial ATP content indicates that the mitochondrial dysfunction is related to MPTP opening due to the oxidative stress resulting from the inhibition of the ETC. This mitochondrial ATP decrease resulted from the failure in the ability of oxidative phosphorylation for ATP production. On the other hand, the fall in the ATP content may exacerbate the ROS formation and lipid peroxidation.46 In addition to enhanced ROS formation, Pb2+ also has the ability to complex with ?SH groups, thus depleting the level of cellular GSH, which plays a critical role in maintaining cellular redox homeostasis.47 Therefore, the decline of reduced GSH content in mitochondria (Fig. 4B) could cause severe deficiency in their defense system against oxidative damage that led to the further increase in lipid peroxidation.48 Lipid peroxidation not only leads to the increase of ROS production but also can damage mitochondrial membrane integrity and open the MPTP. MPTP opening is an important step in both necrosis and apoptosis mechanisms.49In our study, MPTP opening by Pb2+ was doubly confirmed by the MMP collapse (Fig. 5B) which was subsequent to mitochondrial swelling (Fig. 5A). When the MPTP opens, its large conductance can result in rapid swelling due to the osmotic pressure of matrix solutes, the rupture of the outer mitochondrial membrane, and the collapse of the MMP.50MMP is the driving force for H+ efflux and ATP production. Its alteration may change the dynamic behavior of the membrane, which corresponds to the result of the membrane motility change. As shown in Fig. 5C, Pb2+ induced a decrease of the membrane fluidity, thus suggesting that Pb2+ could induce alterations of the mitochondrial inner membrane protein conformation, which confirmed the conclusion that Pb2+ stimulated the opening of MPTP. Then, MPTP opening initiates the mitochondrial inner membrane permeabilization, followed by matrix osmotic swelling, rupture of the mitochondrial outer membrane, and the release of Cyt c (Table 1), which may ultimately activate the caspase family of proteases, the primary trigger leading to the onset of apoptosis.51?53 To investigate the mechanism of MPT induced by Pb2+, the functions of MPT inhibitors and chelators (CsA, ADP, EDTA, EGTA, DMSA and RR) were studied for their influences on mitochondrial swelling in the presence of 80 M Pb2+ (Fig. 5D). CsA demonstrates obvious protective effects, which confirms that the mitochondrial swelling through MPT is induced by Pb2+. The partially protective effect of ADP on the Pb2+-induced mitochondrial swelling indicates that the ANT conformation may be changed.EDTA and EGTA markedly inhibit the Pb2+-induced swelling indicating their possible role in preventing MPT by chelating metal ions. Ca2+ is known to cause MPT by accumulating in the mitochondria matrix and inducing the MMP loss.53 In the presence of RR, Ca2+ is unable to permeate into the matrix and the swelling is protected. In our study, RR totally protects the Pb2+-induced mitochondrial swelling, showing that Pb2+ is also able to influence the Ca2+ regulation. DMSA, a thiol reagent and an antioxidant, can protect the S-sites of the inner membrane components. In this work, we observed that DMSA interacts with Pb2+ due to its active ?SH groups. According to the above discussion, we conclude that free Pb2+ was able to open MPTP, influenced Ca2+ regulation and oxidative damage, which could be proposed and elucidated briefly in Fig. 6.In summary, an overdose of Pb2+ impaired the mitochondrial respiratory chain and inhibited the activity of complexes, especially complex III, which was the reason for increased mitochondrial ROS production. 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