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      Glutamate dehydrogenase as a biomarker for mitotoxicity; insights from furosemide hepatotoxicity in the mouse

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          Abstract

          Glutamate dehydrogenase (GLDH) is a liver-specific biomarker of hepatocellular damage currently undergoing qualification as a drug development tool. Since GLDH is located within the mitochondrial matrix, it has been hypothesized that it might also be useful in assessing mitotoxicity as an initiating event during drug-induced liver injury. According to this hypothesis, hepatocyte death that does not involve primary mitochondrial injury would result in release of intact mitochondria into circulation that could be removed by high speed centrifugation and result in lower GLDH activity measured in spun serum vs un-spun serum. A single prior study in mice has provided some support for this hypothesis. We sought to repeat and extend the findings of this study. Accordingly, mice were treated with the known mitochondrial toxicant, acetaminophen (APAP), or with furosemide (FS), a toxicant believed to cause hepatocyte death through mechanisms not involving mitotoxicity as initiating event. We measured GLDH levels in fresh plasma before and after high speed centrifugation to remove intact mitochondria. We found that both APAP and FS treatments caused substantial hepatocellular necrosis that correlated with plasma alanine aminotransferase (ALT) and GLDH elevations. The plasma GLDH activity in both the APAP- and FS- treated mice was not affected by high-speed centrifugation. Interestingly, the ratio of GLDH:ALT was 5-fold lower during FS compared to APAP hepatotoxicity. Electron microscopy confirmed that both APAP- and FS-treatments had resulted in mitochondrial injury. Mitochondria within vesicles were only observed in the FS-treated mice raising the possibility that mitophagy might account for reduced release of GLDH in the FS-treated mice. Although our results show that plasma GLDH is not clinically useful for evaluating mitotoxicity, the GLDH:ALT ratio as a measure of mitophagy needs to be further studied.

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          Most cited references 21

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          Liver enzyme alteration: a guide for clinicians.

           E Giannini (2005)
          Isolated alterations of biochemical markers of liver damage in a seemingly healthy patient can present a challenge for the clinician. In this review we provide a guide to interpreting alterations to liver enzyme levels. The functional anatomy of the liver and pathophysiology of liver enzyme alteration are briefly reviewed. Using a schematic approach that classifies enzyme alterations as predominantly hepatocellular or predominantly cholestatic, we review abnormal enzymatic activity within the 2 subgroups, the most common causes of enzyme alteration and suggested initial investigations.
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            Blood contains circulating cell‐free respiratory competent mitochondria

            Mitochondria are considered as the power-generating units of the cell due to their key role in energy metabolism and cell signaling. However, mitochondrial components could be found in the extracellular space, as fragments or encapsulated in vesicles. In addition, this intact organelle has been recently reported to be released by platelets exclusively in specific conditions. Here, we demonstrate for the first time, that blood preparation with resting platelets, contains whole functional mitochondria in normal physiological state. Likewise, we show, that normal and tumor cultured cells are able to secrete their mitochondria. Using serial centrifugation or filtration followed by polymerase chain reaction-based methods, and Whole Genome Sequencing, we detect extracellular full-length mitochondrial DNA in particles over 0.22 µm holding specific mitochondrial membrane proteins. We identify these particles as intact cell-free mitochondria using fluorescence-activated cell sorting analysis, fluorescence microscopy, and transmission electron microscopy. Oxygen consumption analysis revealed that these mitochondria are respiratory competent. In view of previously described mitochondrial potential in intercellular transfer, this discovery could greatly widen the scope of cell-cell communication biology. Further steps should be developed to investigate the potential role of mitochondria as a signaling organelle outside the cell and to determine whether these circulating units could be relevant for early detection and prognosis of various diseases.
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              Oxidant stress, mitochondria, and cell death mechanisms in drug-induced liver injury: lessons learned from acetaminophen hepatotoxicity.

              Hepatotoxicity is a serious problem during drug development and for the use of many established drugs. For example, acetaminophen overdose is currently the most frequent cause of acute liver failure in the United States and Great Britain. Evaluation of the mechanisms of drug-induced liver injury indicates that mitochondria are critical targets for drug toxicity, either directly or indirectly through the formation of reactive metabolites. The consequence of these modifications is generally a mitochondrial oxidant stress and peroxynitrite formation, which leads to structural alterations of proteins and mitochondrial DNA and, eventually, to the opening of mitochondrial membrane permeability transition (MPT) pores. MPT pore formation results in a collapse of mitochondrial membrane potential and cessation of adenosine triphosphate synthesis. In addition, the release of intermembrane proteins, such as apoptosis-inducing factor and endonuclease G, and their translocation to the nucleus, leads to nuclear DNA fragmentation. Together, these events trigger necrotic cell death. Alternatively, the release of cytochrome c and other proapoptotic factors from mitochondria can promote caspase activation and apoptotic cell death. Drug toxicity can also induce an inflammatory response with the formation of reactive oxygen species by Kupffer cells and neutrophils. If not properly detoxified, these extracellularly generated oxidants can diffuse into hepatocytes and trigger mitochondrial dysfunction and oxidant stress, which then induces MPT and necrotic cell death. This review addresses the formation of oxidants and the defense mechanisms available for cells and applies this knowledge to better understand mechanisms of drug hepatotoxicity, especially acetaminophen-induced liver injury.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Formal analysisRole: MethodologyRole: Project administrationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: InvestigationRole: MethodologyRole: Project administrationRole: Writing – original draftRole: Writing – review & editing
                Role: InvestigationRole: Project administrationRole: Resources
                Role: Formal analysisRole: InvestigationRole: VisualizationRole: Writing – review & editing
                Role: Formal analysisRole: InvestigationRole: VisualizationRole: Writing – review & editing
                Role: ConceptualizationRole: ResourcesRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS One
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                9 October 2020
                2020
                : 15
                : 10
                Affiliations
                [1 ] Institute for Drug Safety Sciences, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
                [2 ] Division of Pharmacotherapy and Experimental Therapeutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
                [3 ] Pfizer Inc., Groton, Connecticut, United States of America
                University of Navarra School of Medicine and Center for Applied Medical Research (CIMA), SPAIN
                Author notes

                Competing Interests: Authors SJS, GGB, JMK, and JA were employees of Pfizer Inc. at the time this study was completed. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

                [¤]

                Current address: Takeda Pharmaceuticals, Cambridge, Massachusetts, United States of America

                ‡ These authors are joint senior authors on this work.

                Article
                PONE-D-20-22783
                10.1371/journal.pone.0240562
                7546462
                33035276
                © 2020 Church et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Figures: 6, Tables: 0, Pages: 12
                Product
                Funding
                This study was a collaboration between authors PBW and RJC at the University of North Carolina at Chapel Hill and author JA who was at Pfizer when this study was completed. The in-life animal study was performed at UNC (by JSE) while clinical chemistry, histopathology, and electron microscopy were performed at Pfizer (by authors SJS, GGB, and JA). No money was exchanged between UNC and Pfizer for this work; both UNC and Pfizer used internal discretionary funding for this collaboration. Pfizer provided support in the form of salaries for authors (SJS, GGB, JMK, and JA), but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the “authors contributions” section.
                Categories
                Research Article
                Biology and Life Sciences
                Biochemistry
                Bioenergetics
                Energy-Producing Organelles
                Mitochondria
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Energy-Producing Organelles
                Mitochondria
                Biology and Life Sciences
                Anatomy
                Body Fluids
                Blood
                Blood Plasma
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                Body Fluids
                Blood
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                Physiology
                Body Fluids
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                Biochemistry
                Biomarkers
                Research and Analysis Methods
                Separation Processes
                Centrifugation
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Hepatocytes
                Biology and Life Sciences
                Anatomy
                Liver
                Hepatocytes
                Medicine and Health Sciences
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                Liver
                Hepatocytes
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