IntroductionCadmium (Cd) is one of the most dominant heavy metals in the Bohai Sea. Our previous study proved that Cd could induce gill mitochondrial toxicity in marine animals. Herein, we aimed to elucidate the toxicity mechanism of Cd on liver mitochondria, as liver is the main metabolic and detoxification organ and generally rich in mitochondria.MethodsThe mitochondrial responses induced by Cd (5 and 50 μg/L) were characterized by observing mitochondrial morphology, measuring mitochondrial membrane potential (MMP), and proteomic and metabolomic analysis in juvenile olive flounder Paralichthys olivaceus livers.ResultsAfter water-bonre exposure for 14 days, two Cd treatments decreased MMPs significantly and caused ultrastructural-damaged mitochondria in flounder livers. NMR-based metabolomics revealed that Cd exposure mainly altered the abundances of metabolites (ATP, AMP, phosphocholine, lactate and succinate) related to energy metabolism in flounder livers. iTRAQ-based mitochondrial proteomics indicated that 27 differentially expressed proteins (DEPs) were screened out from liver mitochondria after Cd treatments. These proteins were mainly associated with energy metabolism (oxidative phosphorylation (OXPHOS) and tricarboxylic acid (TCA) cycle) and apoptosis.DiscussionThese results indicated that Cd disrupted mitochondrial morphology, energy homeostasis and apoptosis in liver mitochondria in flounder P. olivaceus. This work revealed a comprehensive view on Cd-induced mitochondrial responses in the liver tissues of flounder using an integrated proteomic and metabolomic approach.
Chlorella is a unicellular spherical green microalga with alternate colors from blue green to yellowish or red due to different components of innate pigments. Light and salinity are two important environmental factors in Chlorella culture. Light conditions directly affect the growth and biochemical composition of microalgae, while salinity change could influence the pigment composition of Chlorella. Therefore, it has crucial research significance to monitor the response of Chlorella to salinity stress under different light conditions. Recently, Fluorescence Lifetime Imaging Microscopy (FLIM) technology has been widely applied into biological fields, providing fluorescence lifetime values for quantitative analysis. Here, FLIM method was used to observe the autofluorescence of a freshwater microalga, Chlorella sp.. Chlorella cells were treated with a series of salinity concentrations (control sample in normal culture medium, 3S sample with an additional 3× salinity, 7S sample with an additional 7× salinity, respectively) under light (12 h/12 h light/dark cycles) or dark (0 h/24 h light/dark cycles) treatments. After one day, images of the microalgae cells from each group were obtained with FLIM system, followed by an analysis with SPCImage software. The results showed that 3× salinity condition had little effect on Chlorella in both light/dark conditions, suggesting the adaptive capacity of Chlorella to seawater salinity. By contrast, the mean fluorescence lifetime values in 7S samples under light conditions were significantly decreased compared to that of the control. Interestingly, similar lifetime values were observed in 7S samples and the control samples under dark conditions, which indicated a potential high salinity resistance induced by different light/dark conditions. In conclusion, FLIM could work as a fast evaluation method of the physiological status of living Chlorella sp. under different culture conditions in a quantitative way.
Inorganic arsenic (iAs) is one of the most toxic metalloids which could accumulate in marine species, especially in clams, causing serious ecological risk. Marine clams accumulate high level of iAs in different tissues. Currently, fluorescence lifetime microscopy (FLIM) technique has provided quantitative information in biochemical diagnosis. In this study, we applied FLIM method into analyzing Hematoxylin and eosin(H&E) stained sections of arsenic exposed Ruditapes philippinarum. The clams were exposed under different concentrations of As(Ⅲ) and As(V) for thirty days. Fluorescent images of the H&E stained hepatopancreas tissue were obtained with FLIM system, followed with data analysis for fluorescence lifetime values. The average fluorescence lifetime of sections in the control group was around 250 ps. The average lifetime value in the As(V) group was slightly increased to around 280-300 ps. The average lifetime value in the As(III) group achieved a significant increase to around 340 ps. These results suggested a higher extent of structural change in As(Ⅲ) exposed group than As(V) group. As a result, this work has provided quantitative evaluation standard for the toxicity of marine mollusk based on fluorescence lifetime imaging method.
Chlorella is a single-celled blue-green spherical microalga, whose color could change from green to red or yellowish due to the components of different types of innate pigments. Salinity change is one important environmental stressor that may influence the pigment composition of Chlorella. Therefore, it is necessary to monitor the salinity stress on Chlorella in a real-time mode. Recently, fluorescence lifetime imaging microscopy (FLIM) technology has been widely applied into biological fields, which could provide fluorescence lifetime values for quantitative analysis. Here, we used FLIM method to investigate a freshwater microalga, Chlorella sp. based on its autofluorescence. Chlorella cells were treated with a series of salinity concentrations (control sample in normal culture medium, 3S sample with an additional 3× salinity, 7S sample with an additional 7× salinity, respectively) for one day. Then images of the microalgae cells from each group were obtained with FLIM system and analyzed with SPCImage software, providing the fluorescence lifetime data. The results of fluorescence lifetime data showed that 3× salinity condition had little effect on Chlorella, which indicated that Chlorella had a strong adaptive capacity in environments close to seawater salinity. However, the significant left shift of lifetime distribution peak and decreased mean lifetime values were observed in 7S samples compared with the control. In conclusion, FLIM method has shown great potential as a fast identification method of living Chlorella sp. under high salinity conditions in a quantitative and non-invasive way.
With the rapid development of modern industry, heavy metal pollution is becoming one of the most severe marine environmental problems among the world. Mussels are considered as suitable models to monitor heavy metal pollutions.The hematoxylin and eosin (H&E) sections of mussels could be observed with fluorescence lifetime imaging microscopy (FLIM) method, generating quantitative data of images for improved analysis with higher accuracy. In this study, we used FLIM method to investigate the fluorescence lifetime of digestive glands of adult mussels Mytilus galloprovincialis treated with two environmentally relevant concentrations (5 μg/L and 50 μg/L) of cadmium (Cd) for 14 days. After exposure, the H&E stained sections of the digestive glands were observed with FLIM system. The images were analyzed with SPCImage software, providing both the structural images with fluorescence intensity information and the pseudo-color images with fluorescence lifetime information. The fluorescence lifetime values were presented as total τ, which could be divided into τ1 (the shorter part) and τ2 (the longer part), respectively. Results showed that the τ2 values in 50 μg/L Cd treatments were significantly lower than those of the control group, suggesting that the high concentration of Cd treatment has a high impact on the digestive glands of M. galloprovincialis. The significance of the fluorescence lifetime data of mussel digestive glands induced by Cd at a high concentration has proved the sensitivity of FLIM method to distinguish the polluted mussels from the healthy individuals. In conclusion, FLIM has high potential in further applications of marine environment pollution monitoring.
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