| 其他摘要 | Mitochondrion is the center of energy metabolism in eukaryon, which generates most of the energy for normal physiological activities. Apart from the vital role in energy metabolism, mitochondria also participate in the regulation of cell proliferation and differentiation, redox homeostasis, calcium homeostasis, endocrine, apoptosis, etc. Our previous studies showed that cadmium (Cd) and arsenic (As) could significantly affect the energy metabolism process in marine animals, such as oysters, clams, mussels and fish. Proteomic analysis indicated that 20% - 33% of the differentially expressed proteins (DEPs) in juvenile olive flounder Paralichthys olivaceus induced by Cd and As were related to energy metabolism. As it is known, energy metabolism plays vital roles in growth and survival, which is the primary basis of proliferation and differentiation, immune responses, osmoregulation, reproduction and development and other processes. Mitochondrion is susceptible and may be the main bio-target of Cd and As, playing an essential role in response to the internal or external stress. However, mitochondrial toxicities of Cd and As have not been fully investigated in marine organisms. In this work, metabolomics, proteomics and traditional ecotoxicological methods were integrated to systematically elucidate the mitochondrial toxicity and its mechanisms in gill and liver tissues of juvenile olive flounder P. olivaceus induced by two environmental related concentrations (5 μg/L and 50 μg/L) of Cd and As(Ⅴ) for 14 days, aiming to construct the mitochondrial responsive pathways and providing theoretical basis for biological monitoring and risk assessment of heavy metal pollutions (Cd and As) in the Bohai Sea. The main results were as follows:
1. Cd-induced mitochondrial toxicity and its mechanism in juvenile flounder
After exposed to Cd for 14 days, the mitochondrial toxicity of Cd was characterized by the mitochondrial morphology observation, mitochondrial membrane potential detection, as well as metabolomic and proteomic analysis in juvenile flounder. At the subcellular levels, transmission electron microscopy (TEM) observations showed that 5 μg/L and 50 μg/L Cd induced mitochondrial damages in juvenile flounder gills, mainly reflected by injured or shed mitochondrial membrane. Compared with the control, dose-dependent increases of mitochondrial density (P < 0.01) and decreases of mitochondrial area index (P < 0.01) were induced after Cd treatments, indicating that Cd could not only cause mitochondrial damages but also induce more and smaller gill mitochondria. Mitochondrial membrane potential, one of the key factors to maintain mitochondrial activity and function, was detected to evaluate the toxic effects of Cd on mitochondrial function. Both Cd treatments (5 μg/L and 50 μg/L) significantly reduced membrane potentials (P < 0.05) of gill mitochondria in a dose-dependent manner. Metabolomic analysis revealed that metabolites related to energy metabolism, such as ATP, AMP and creatine phosphate, were significantly altered in gills after Cd treatments. iTRAQ-based mitochondrial proteomic analysis showed that Cd induced 74 and 118 DEPs in 5 μg/L and 50 μg/L Cd-treated juvenile flounder gills, respectively. Most of them were up-regulated and mainly involved in energy metabolism processes such as tricarboxylic acid (TCA) cycle and oxidative phosphorylation. Further analysis on the mechanisms of Cd-induced mitochondrial toxicity in juvenile flounder gills suggested that the TCA cycle and oxidative phosphorylation was enhanced to promote ATP production in gill mitochondria to meet the energy demand after Cd treatments. The DEPs involved in mitochondrial morphology and import (mitochondrial contact site and cristae organizing system complex, mitochondrial import inner membrane translocase, etc.) were up-regulated to enhance mitochondrial protein import and mitochondrial network stability in juvenile flounder gills after Cd exposure. For stress resistance and apoptosis, ROS accumulation and Ca2+ overload were induced by up-regulated reactive oxygen species modulator 1 and protein NipSnap homolog 2. Several altered apoptosis-related proteins, such as voltage-dependent anion-selective channel protein, peptidyl-prolyl isomerase F, ADP/ATP translocase 2, and Bcl2-associated agonist of cell death, facilitated the opening of mitochondrial permeability transition pore and the release of apoptosis-inducing factor 1, eventually caused apoptosis. In addition, mitochondrial responses to Cd in juvenile flounder gills were also related to cholesterol metabolism.
Cd induced mitochondrial injuries, increased mitochondrial density and mitochondrial area index (P < 0.05), accompanied by a large number of lipid droplets in juvenile flounder livers, indicating that Cd could not only damage mitochondrial morphology but also lead to metabolic dysfunction in liver mitochondria. Consistently, the decreased mitochondrial membrane potentials (P < 0.01) also indicated that Cd disturbed the normal function of liver mitochondria. At the molecular levels, like the metabolomic results of gill samples, differentially altered metabolites related to energy metabolism were identified in livers. However, some of them such as ATP, AMP and choline phosphate showed opposite alteration trends in 5 μg/L and 50 μg/L Cd-treated groups, suggesting the different responsive patterns of energy metabolism in founder gill and liver tissues after Cd exposure. Proteomic analysis showed that Cd induced 28 DEPs in liver mitochondria. These DEPs were mainly involved in energy metabolism, stress resistance and apoptosis. There were 14 and 19 DEPs in 5 μg/L and 50 μg/L Cd treatments, respectively. Compared with the gill mitochondria, Cd induced less DEPs in liver mitochondria, and most of them were down-regulated. Further analysis on the mechanisms of Cd-induced mitochondrial toxicity in juvenile flounder livers confirmed that Cd enhanced anaerobic metabolism, suppressed mitochondrial import, and disturbed energy production by regulating TCA cycle and inhibiting the electron transport in oxidative phosphorylation. Besides, down-regulated protein phosphatase 1K and calcium uptake protein 1 meant the alteration of mitochondrial permeability transition pore and Ca2+ uptake in livers of Cd-exposed juvenile flounders. The differentially expressed pro-apoptotic protein (second mitochondrial derived activator of caspase homolog) and anti-apoptosis protein (ATP-dependent RNA helicas and ATP-dependent zinc metalloprotease) illustrated that Cd disturbed mitochondrial-dependent apoptosis pathway in juvenile flounder livers.
2. As(Ⅴ)-induced mitochondrial toxicity and its mechanism in juvenile flounder
After exposure for 14 days, ultrastructural damages of gill mitochondria were induced by 5 μg/L and 50 μg/L As(Ⅴ) , indicated by loose, fragmented and detached mitochondrial cristae. Besides, mitochondrial density was significantly increased (P < 0.01) with the increase of As(Ⅴ) concentrations for exposures, while mitochondrial area index decreased, indicating that As(Ⅴ) induced more and smaller gill mitochondria. Then the toxic effects of As(Ⅴ) on mitochondrial functions were characterized by mitochondrial membrane potential detection as well as metabolomic and proteomic analysis. Compared with the control, mitochondrial membrane potentials were significantly decreased (P < 0.05) in As(Ⅴ)-treated juvenile flounder gills. Metabolites related to anaerobic respiration and energy metabolism, such as ATP, AMP, lactate and glucose, were altered in gill samples after As(Ⅴ) exposure. Proteomic analysis discovered 83 DEPs in gill mitochondria after As(Ⅴ) exposures, which were mainly related to mitochondrial morphology and import, energy metabolism, and stress and apoptosis. There were 60 and 68 DEPs in 5 μg/L and 50 μg/L As(Ⅴ)-treated groups, respectively, most of which were up-regulated DEPs. Further analysis on the mechanisms of As(Ⅴ)-induced mitochondrial toxicity in juvenile flounder gills illustrated that As(Ⅴ) damaged mitochondria and enhanced mitochondrial protein import by regulating mitochondrial contact site and cristae organizing system complex, inorganic pyrophosphatase 2, etc. The anaerobic metabolism, TCA cycle and oxidative phosphorylation electron transfer were promoted to enhance energy production in flounder gill mitochondria under As(Ⅴ) stress. Moreover, the mitochondrial membrane permeability was increased due to differentially expressed voltage-dependent anion-selective channel protein, ADP/ATP translocase and phosphate carrier protein. Meanwhile, altered apoptosis-associated proteins (Bcl2-associated agonist of cell death, ATP-dependent RNA helicase and serine protease, etc.) might inhibit the mitochondrial-dependent apoptosis in juvenile flounder gills.
As(Ⅴ) treatments injured mitochondrial cristae and increased the mitochondrial density (P < 0.01) and in juvenile flounder livers in a dose-dependent manner. The mitochondrial area indexes were also increased, but there was only significant difference between control and 5 μg/L As(Ⅴ)-treated group (P < 0.01), indicating that 5 μg/L As(Ⅴ) induced more mitochondria and 50 μg/L As(Ⅴ) caused more and smaller liver mitochondria. Besides, liver mitochondrial membrane potentials were significantly decreased (P < 0.05) in As(Ⅴ)-treated groups. Metabolomic analysis showed that differentially altered metabolites such as ATP, AMP, lactate and glucose were only identified in 50 μg/L As(Ⅴ) treatment, suggesting that 50 μg/L As(Ⅴ) had stronger effects on liver anaerobic respiration and energy metabolism than 5 μg/L As(Ⅴ). Proteomic analysis identified 40 DEPs in liver mitochondria after As(Ⅴ) exposures, which were basically involved in energy metabolism and stress and apoptosis, including 5 and 35 DEPs in these two As(Ⅴ)-treated groups, respectively. Further analysis on the mechanisms of As(Ⅴ)-induced mitochondrial toxicity in juvenile flounder livers exhibited that As(Ⅴ) enhanced liver anaerobic metabolism and TCA cycle and inhibited ATP production in oxidative phosphorylation, resulting in energy metabolism disturbance. For stress resistance and apoptosis, As(Ⅴ) exposure increased mitochondrial membrane permeability and disturbed mitochondrial-dependent apoptosis in juvenile flounder livers by up-regulation of ADP/ATP translocase 3, complement component 1 Q subcomponent-binding protein and presenilins-associated rhomboid-like protein. On the other hand, liver mitochondria maintained ROS homeostasis by up-regulating methionine-R-sulfoxide reductase B2 and thioredoxin 2 to reduce mitochondrial damages induced by As(Ⅴ) treatments. Moreover, mitochondrial responses to As(Ⅴ) in juvenile flounder livers were also involved in protein translation and modification, and vitamin D synthesis.
3. Comparison of the toxic mechanisms of Cd and As(V) on juvenile flounder mitochondria
There existed differences of toxicity mechanisms between Cd and As(V) on flounder mitochondria. Both Cd and As(V) increased mitochondria number, caused mitochondrial damages and decreased mitochondrial membrane potentials. Meanwhile, they promoted TCA cycle and oxidative phosphorylation. In addition, Cd significantly increased the morphological stability and protein import of gill mitochondria and induced mitochondrial-dependent apoptosis. However, As(V) significantly down-regulated morphological stability of gill mitochondria but enhanced mitochondrial protein import, promoted anaerobic respiration, and inhibited mitochondrial-dependent apoptosis. Both Cd and As(V) facilitated liver anaerobic metabolism and disturbed mitochondrial-dependent apoptosis. Cd inhibited mitochondrial protein import and oxidative phosphorylation of liver mitochondria and disturbed TCA cycle, while As(V) enhanced TCA cycle and ROS homeostasis and decreased ATP production. |
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