Reactive oxygen species in the normal and acutely injured liver

Livers generate low levels of reactive oxygen species (ROS), especially superoxide in the mitochondria, and hydrogen peroxide as a normal function of various oxidases [1Jaeschke H. Antioxidant defense mechanisms.in: Comprehensive toxicology. vol. 9. Academic Press, Oxford2010: 319-337Google Scholar]. The large number of mitochondria and their capacity to leak electrons from complex I and III of the electron transport chain make them quantitatively the most important intracellular source of ROS [1Jaeschke H. Antioxidant defense mechanisms.in: Comprehensive toxicology. vol. 9. Academic Press, Oxford2010: 319-337Google Scholar]. ROS formation is dangerous for cells due to the presence of polyunsaturated fatty acids in cellular membranes, the substantial number of unprotected protein sulfhydryl groups and DNA bases. Therefore, cells had to develop sophisticated defense systems. Each liver cell expresses superoxide dismutases (SOD1 in the cytosol; SOD2 in mitochondria), glutathione peroxidases (cytosol and mitochondria), catalase (peroxisomes), thioredoxins (Trx1 in cytosol; Trx2 in mitochondria) and peroxiredoxins (Prx-I, -II, -VI in the cytosol; Prx-III, -V in mitochondria) [1Jaeschke H. Antioxidant defense mechanisms.in: Comprehensive toxicology. vol. 9. Academic Press, Oxford2010: 319-337Google Scholar] (Fig. 1A). In addition, liver cells contain mM concentrations of glutathione in all cellular compartments, have radical chain-breaking antioxidants (vitamin E) in cell membranes, and keep redox-active iron tightly bound to storage or transport proteins [1Jaeschke H. Antioxidant defense mechanisms.in: Comprehensive toxicology. vol. 9. Academic Press, Oxford2010: 319-337Google Scholar]. Because of this multi-layer defense system against ROS, liver cells and especially hepatocytes, have a substantial capacity to metabolize and effectively detoxify ROS and repair oxidant damage. Therefore, under realistic in vivo conditions, catastrophic free radical events such as lipid peroxidation are rarely the cause of cell death [2Jaeschke H. Reactive oxygen and mechanisms of inflammatory liver injury: present concepts.J Gastroenterol Hepatol. 2011; 26: 173-179Crossref PubMed Scopus (395) Google Scholar]. Instead, ROS generally cause disturbances to cellular homeostasis and, if not effectively counteracted, this can lead to cell death.An example where the combination of increased ROS formation and impaired defense systems causes cell death is acetaminophen (APAP) overdose, which is the leading cause of acute liver failure in Western countries [3Jaeschke H. Bajt M.L. Intracellular signaling mechanisms of acetaminophen-induced liver cell death.Toxicol Sci. 2006; 89: 31-41Crossref PubMed Scopus (429) Google Scholar]. The reactive metabolite of APAP depletes glutathione (GSH) in the cytosol and in mitochondria and binds to cellular proteins, which causes formation of ROS and peroxynitrite inside mitochondria [3Jaeschke H. Bajt M.L. Intracellular signaling mechanisms of acetaminophen-induced liver cell death.Toxicol Sci. 2006; 89: 31-41Crossref PubMed Scopus (429) Google Scholar] (Fig. 1A). In the absence of mitochondrial GSH, the mitochondrial oxidant stress triggers the opening of the mitochondrial permeability transition (MPT) pore, which causes the collapse of the membrane potential and cessation of ATP synthesis. In addition, mitochondrial intermembrane proteins endonuclease G and apoptosis-inducing factor translocate to the nucleus and cause DNA fragmentation [3Jaeschke H. Bajt M.L. Intracellular signaling mechanisms of acetaminophen-induced liver cell death.Toxicol Sci. 2006; 89: 31-41Crossref PubMed Scopus (429) Google Scholar]. The critical role of the impaired mitochondrial antioxidant defense system is clearly demonstrated by the profound hepatoprotection if ROS and peroxynitrite are effectively scavenged by accelerated recovery of mitochondrial GSH [4Saito C. Zwingmann C. Jaeschke H. Novel mechanisms of protection against acetaminophen hepatotoxicity in mice by glutathione and N-acetylcysteine.Hepatology. 2010; 51: 246-254Crossref PubMed Scopus (301) Google Scholar].A critical role of ROS in the pathophysiology is also established in acute inflammatory conditions as observed after hepatic ischemia–reperfusion injury (liver resections, transplantation), obstructive cholestasis, or endotoxemia/sepsis [5Jaeschke H. Mechanisms of liver injury. II. Mechanisms of neutrophil-induced liver cell injury during hepatic ischemia-reperfusion and other acute inflammatory conditions.Am J Physiol Gastrointest Liver Physiol. 2006; 290: G1083-G1088Crossref PubMed Scopus (394) Google Scholar]. Tissue trauma (ischemic damage) leads to release of damage-associated molecular patterns (DAMPs) (Fig. 1B), which can activate the complement cascade and are direct ligands of toll-like receptors 6Bianchi M.E. HMGB1 loves company.J Leukoc Biol. 2009; 86: 573-576Crossref PubMed Scopus (320) Google Scholar, 7Zhang Q. Raoof M. Chen Y. Sumi Y. Sursal T. Junger W. et al.Circulating mitochondrial DAMPs cause inflammatory responses to injury.Nature. 2010; 464: 104-107Crossref PubMed Scopus (2406) Google Scholar. DAMPS include molecules such as high mobility group box protein (HMGB1), heat shock proteins (HSPs), formyl peptides, and mitochondrial and nuclear DNA fragments 6Bianchi M.E. HMGB1 loves company.J Leukoc Biol. 2009; 86: 573-576Crossref PubMed Scopus (320) Google Scholar, 7Zhang Q. Raoof M. Chen Y. Sumi Y. Sursal T. Junger W. et al.Circulating mitochondrial DAMPs cause inflammatory responses to injury.Nature. 2010; 464: 104-107Crossref PubMed Scopus (2406) Google Scholar. Combinations of DAMPs are the most potent inflammagens [6Bianchi M.E. HMGB1 loves company.J Leukoc Biol. 2009; 86: 573-576Crossref PubMed Scopus (320) Google Scholar]. Activated complement fragments prime and activate macrophages for ROS formation through NADPH oxidase. ROS generated in the vascular space of the liver, mainly superoxide and hydrogen peroxide, can be detoxified in the extracellular space by GSH released from hepatocytes. However, excessive ROS formation results in diffusion of the ROS into hepatocytes and formation of an intracellular oxidant stress that can cause cell injury through mitochondrial dysfunction [8Bilzer M. Jaeschke H. Vollmar A.M. Paumgartner G. Gerbes A.L. Prevention of Kupffer cell-induced oxidant injury in rat liver by atrial natriuretic peptide.Am J Physiol. 1999; 276: G1137-G1144PubMed Google Scholar] (Fig. 1B).DAMPs or pathogen-associated molecular patterns (PAMPs) induce cytokine and chemokine formation in macrophages (Kupffer cells) and other innate immune cells through toll-like receptors 5Jaeschke H. Mechanisms of liver injury. II. Mechanisms of neutrophil-induced liver cell injury during hepatic ischemia-reperfusion and other acute inflammatory conditions.Am J Physiol Gastrointest Liver Physiol. 2006; 290: G1083-G1088Crossref PubMed Scopus (394) Google Scholar, 6Bianchi M.E. HMGB1 loves company.J Leukoc Biol. 2009; 86: 573-576Crossref PubMed Scopus (320) Google Scholar, 7Zhang Q. Raoof M. Chen Y. Sumi Y. Sursal T. Junger W. et al.Circulating mitochondrial DAMPs cause inflammatory responses to injury.Nature. 2010; 464: 104-107Crossref PubMed Scopus (2406) Google Scholar. This triggers activation and recruitment of inflammatory cells, especially neutrophils. Although these cells can be primed for ROS formation, in general they do not produce ROS in the vasculature [5Jaeschke H. Mechanisms of liver injury. II. Mechanisms of neutrophil-induced liver cell injury during hepatic ischemia-reperfusion and other acute inflammatory conditions.Am J Physiol Gastrointest Liver Physiol. 2006; 290: G1083-G1088Crossref PubMed Scopus (394) Google Scholar]. After receiving a chemotactic signal from the parenchyma, neutrophils transmigrate and adhere to targets, which lead to full activation and a long-lasting adherence-dependent oxidant stress in close proximity to hepatocytes (Fig. 1C). Although both resident macrophages and neutrophils are generating superoxide and hydrogen peroxide through NADPH oxidase, neutrophils also contain myeloperoxidase, which can form the potent oxidant and chlorinating species hypochlorous acid [1Jaeschke H. Antioxidant defense mechanisms.in: Comprehensive toxicology. vol. 9. Academic Press, Oxford2010: 319-337Google Scholar]. There is evidence that both hydrogen peroxide and hypochlorous acid from neutrophils diffuse into the target cell and generate an intracellular oxidant stress [5Jaeschke H. Mechanisms of liver injury. II. Mechanisms of neutrophil-induced liver cell injury during hepatic ischemia-reperfusion and other acute inflammatory conditions.Am J Physiol Gastrointest Liver Physiol. 2006; 290: G1083-G1088Crossref PubMed Scopus (394) Google Scholar]. It is important to recognize that neutrophils do not attack healthy cells and healthy cells are not killed accidentally by oxidant stress as innocent bystanders [5Jaeschke H. Mechanisms of liver injury. II. Mechanisms of neutrophil-induced liver cell injury during hepatic ischemia-reperfusion and other acute inflammatory conditions.Am J Physiol Gastrointest Liver Physiol. 2006; 290: G1083-G1088Crossref PubMed Scopus (394) Google Scholar]. In contrast, neutrophils target stressed cells where ROS can lead to lysosomal iron mobilization and translocation to the mitochondria, which triggers a mitochondrial oxidant stress, the MPT and eventually necrotic cell death [9Uchiyama A. Kim J.S. Kon K. Jaeschke H. Ikejima K. Watanabe S. et al.Translocation of iron from lysosomes into mitochondria is a key event during oxidative stress-induced hepatocellular injury.Hepatology. 2008; 48: 1644-1654Crossref PubMed Scopus (116) Google Scholar]. In general, even an excess oxidant stress in vivo does not induce apoptosis as frequently seen in vitro but only leads to necrotic cell death when intracellular defense systems are exhausted [10Hong J.Y. Lebofsky M. Farhood A. Jaeschke H. Oxidant stress-induced liver injury in vivo: role of apoptosis, oncotic necrosis, and c-Jun NH2-terminal kinase activation.Am J Physiol Gastrointest Liver Physiol. 2009; 296: G572-G581Crossref PubMed Scopus (57) Google Scholar] (Fig. 1A). This is the reason why during inflammatory liver injury phagocyte-derived ROS primarily induce oncotic necrosis [2Jaeschke H. Reactive oxygen and mechanisms of inflammatory liver injury: present concepts.J Gastroenterol Hepatol. 2011; 26: 173-179Crossref PubMed Scopus (395) Google Scholar] (Fig. 1B and C).DisclosureThe underlying research reported in the study was funded by the NIH Institutes of Health. Livers generate low levels of reactive oxygen species (ROS), especially superoxide in the mitochondria, and hydrogen peroxide as a normal function of various oxidases [1Jaeschke H. Antioxidant defense mechanisms.in: Comprehensive toxicology. vol. 9. Academic Press, Oxford2010: 319-337Google Scholar]. The large number of mitochondria and their capacity to leak electrons from complex I and III of the electron transport chain make them quantitatively the most important intracellular source of ROS [1Jaeschke H. Antioxidant defense mechanisms.in: Comprehensive toxicology. vol. 9. Academic Press, Oxford2010: 319-337Google Scholar]. ROS formation is dangerous for cells due to the presence of polyunsaturated fatty acids in cellular membranes, the substantial number of unprotected protein sulfhydryl groups and DNA bases. Therefore, cells had to develop sophisticated defense systems. Each liver cell expresses superoxide dismutases (SOD1 in the cytosol; SOD2 in mitochondria), glutathione peroxidases (cytosol and mitochondria), catalase (peroxisomes), thioredoxins (Trx1 in cytosol; Trx2 in mitochondria) and peroxiredoxins (Prx-I, -II, -VI in the cytosol; Prx-III, -V in mitochondria) [1Jaeschke H. Antioxidant defense mechanisms.in: Comprehensive toxicology. vol. 9. Academic Press, Oxford2010: 319-337Google Scholar] (Fig. 1A). In addition, liver cells contain mM concentrations of glutathione in all cellular compartments, have radical chain-breaking antioxidants (vitamin E) in cell membranes, and keep redox-active iron tightly bound to storage or transport proteins [1Jaeschke H. Antioxidant defense mechanisms.in: Comprehensive toxicology. vol. 9. Academic Press, Oxford2010: 319-337Google Scholar]. Because of this multi-layer defense system against ROS, liver cells and especially hepatocytes, have a substantial capacity to metabolize and effectively detoxify ROS and repair oxidant damage. Therefore, under realistic in vivo conditions, catastrophic free radical events such as lipid peroxidation are rarely the cause of cell death [2Jaeschke H. Reactive oxygen and mechanisms of inflammatory liver injury: present concepts.J Gastroenterol Hepatol. 2011; 26: 173-179Crossref PubMed Scopus (395) Google Scholar]. Instead, ROS generally cause disturbances to cellular homeostasis and, if not effectively counteracted, this can lead to cell death. An example where the combination of increased ROS formation and impaired defense systems causes cell death is acetaminophen (APAP) overdose, which is the leading cause of acute liver failure in Western countries [3Jaeschke H. Bajt M.L. Intracellular signaling mechanisms of acetaminophen-induced liver cell death.Toxicol Sci. 2006; 89: 31-41Crossref PubMed Scopus (429) Google Scholar]. The reactive metabolite of APAP depletes glutathione (GSH) in the cytosol and in mitochondria and binds to cellular proteins, which causes formation of ROS and peroxynitrite inside mitochondria [3Jaeschke H. Bajt M.L. Intracellular signaling mechanisms of acetaminophen-induced liver cell death.Toxicol Sci. 2006; 89: 31-41Crossref PubMed Scopus (429) Google Scholar] (Fig. 1A). In the absence of mitochondrial GSH, the mitochondrial oxidant stress triggers the opening of the mitochondrial permeability transition (MPT) pore, which causes the collapse of the membrane potential and cessation of ATP synthesis. In addition, mitochondrial intermembrane proteins endonuclease G and apoptosis-inducing factor translocate to the nucleus and cause DNA fragmentation [3Jaeschke H. Bajt M.L. Intracellular signaling mechanisms of acetaminophen-induced liver cell death.Toxicol Sci. 2006; 89: 31-41Crossref PubMed Scopus (429) Google Scholar]. The critical role of the impaired mitochondrial antioxidant defense system is clearly demonstrated by the profound hepatoprotection if ROS and peroxynitrite are effectively scavenged by accelerated recovery of mitochondrial GSH [4Saito C. Zwingmann C. Jaeschke H. Novel mechanisms of protection against acetaminophen hepatotoxicity in mice by glutathione and N-acetylcysteine.Hepatology. 2010; 51: 246-254Crossref PubMed Scopus (301) Google Scholar]. A critical role of ROS in the pathophysiology is also established in acute inflammatory conditions as observed after hepatic ischemia–reperfusion injury (liver resections, transplantation), obstructive cholestasis, or endotoxemia/sepsis [5Jaeschke H. Mechanisms of liver injury. II. Mechanisms of neutrophil-induced liver cell injury during hepatic ischemia-reperfusion and other acute inflammatory conditions.Am J Physiol Gastrointest Liver Physiol. 2006; 290: G1083-G1088Crossref PubMed Scopus (394) Google Scholar]. Tissue trauma (ischemic damage) leads to release of damage-associated molecular patterns (DAMPs) (Fig. 1B), which can activate the complement cascade and are direct ligands of toll-like receptors 6Bianchi M.E. HMGB1 loves company.J Leukoc Biol. 2009; 86: 573-576Crossref PubMed Scopus (320) Google Scholar, 7Zhang Q. Raoof M. Chen Y. Sumi Y. Sursal T. Junger W. et al.Circulating mitochondrial DAMPs cause inflammatory responses to injury.Nature. 2010; 464: 104-107Crossref PubMed Scopus (2406) Google Scholar. DAMPS include molecules such as high mobility group box protein (HMGB1), heat shock proteins (HSPs), formyl peptides, and mitochondrial and nuclear DNA fragments 6Bianchi M.E. HMGB1 loves company.J Leukoc Biol. 2009; 86: 573-576Crossref PubMed Scopus (320) Google Scholar, 7Zhang Q. Raoof M. Chen Y. Sumi Y. Sursal T. Junger W. et al.Circulating mitochondrial DAMPs cause inflammatory responses to injury.Nature. 2010; 464: 104-107Crossref PubMed Scopus (2406) Google Scholar. Combinations of DAMPs are the most potent inflammagens [6Bianchi M.E. HMGB1 loves company.J Leukoc Biol. 2009; 86: 573-576Crossref PubMed Scopus (320) Google Scholar]. Activated complement fragments prime and activate macrophages for ROS formation through NADPH oxidase. ROS generated in the vascular space of the liver, mainly superoxide and hydrogen peroxide, can be detoxified in the extracellular space by GSH released from hepatocytes. However, excessive ROS formation results in diffusion of the ROS into hepatocytes and formation of an intracellular oxidant stress that can cause cell injury through mitochondrial dysfunction [8Bilzer M. Jaeschke H. Vollmar A.M. Paumgartner G. Gerbes A.L. Prevention of Kupffer cell-induced oxidant injury in rat liver by atrial natriuretic peptide.Am J Physiol. 1999; 276: G1137-G1144PubMed Google Scholar] (Fig. 1B). DAMPs or pathogen-associated molecular patterns (PAMPs) induce cytokine and chemokine formation in macrophages (Kupffer cells) and other innate immune cells through toll-like receptors 5Jaeschke H. Mechanisms of liver injury. II. Mechanisms of neutrophil-induced liver cell injury during hepatic ischemia-reperfusion and other acute inflammatory conditions.Am J Physiol Gastrointest Liver Physiol. 2006; 290: G1083-G1088Crossref PubMed Scopus (394) Google Scholar, 6Bianchi M.E. HMGB1 loves company.J Leukoc Biol. 2009; 86: 573-576Crossref PubMed Scopus (320) Google Scholar, 7Zhang Q. Raoof M. Chen Y. Sumi Y. Sursal T. Junger W. et al.Circulating mitochondrial DAMPs cause inflammatory responses to injury.Nature. 2010; 464: 104-107Crossref PubMed Scopus (2406) Google Scholar. This triggers activation and recruitment of inflammatory cells, especially neutrophils. Although these cells can be primed for ROS formation, in general they do not produce ROS in the vasculature [5Jaeschke H. Mechanisms of liver injury. II. Mechanisms of neutrophil-induced liver cell injury during hepatic ischemia-reperfusion and other acute inflammatory conditions.Am J Physiol Gastrointest Liver Physiol. 2006; 290: G1083-G1088Crossref PubMed Scopus (394) Google Scholar]. After receiving a chemotactic signal from the parenchyma, neutrophils transmigrate and adhere to targets, which lead to full activation and a long-lasting adherence-dependent oxidant stress in close proximity to hepatocytes (Fig. 1C). Although both resident macrophages and neutrophils are generating superoxide and hydrogen peroxide through NADPH oxidase, neutrophils also contain myeloperoxidase, which can form the potent oxidant and chlorinating species hypochlorous acid [1Jaeschke H. Antioxidant defense mechanisms.in: Comprehensive toxicology. vol. 9. Academic Press, Oxford2010: 319-337Google Scholar]. There is evidence that both hydrogen peroxide and hypochlorous acid from neutrophils diffuse into the target cell and generate an intracellular oxidant stress [5Jaeschke H. Mechanisms of liver injury. II. Mechanisms of neutrophil-induced liver cell injury during hepatic ischemia-reperfusion and other acute inflammatory conditions.Am J Physiol Gastrointest Liver Physiol. 2006; 290: G1083-G1088Crossref PubMed Scopus (394) Google Scholar]. It is important to recognize that neutrophils do not attack healthy cells and healthy cells are not killed accidentally by oxidant stress as innocent bystanders [5Jaeschke H. Mechanisms of liver injury. II. Mechanisms of neutrophil-induced liver cell injury during hepatic ischemia-reperfusion and other acute inflammatory conditions.Am J Physiol Gastrointest Liver Physiol. 2006; 290: G1083-G1088Crossref PubMed Scopus (394) Google Scholar]. In contrast, neutrophils target stressed cells where ROS can lead to lysosomal iron mobilization and translocation to the mitochondria, which triggers a mitochondrial oxidant stress, the MPT and eventually necrotic cell death [9Uchiyama A. Kim J.S. Kon K. Jaeschke H. Ikejima K. Watanabe S. et al.Translocation of iron from lysosomes into mitochondria is a key event during oxidative stress-induced hepatocellular injury.Hepatology. 2008; 48: 1644-1654Crossref PubMed Scopus (116) Google Scholar]. In general, even an excess oxidant stress in vivo does not induce apoptosis as frequently seen in vitro but only leads to necrotic cell death when intracellular defense systems are exhausted [10Hong J.Y. Lebofsky M. Farhood A. Jaeschke H. Oxidant stress-induced liver injury in vivo: role of apoptosis, oncotic necrosis, and c-Jun NH2-terminal kinase activation.Am J Physiol Gastrointest Liver Physiol. 2009; 296: G572-G581Crossref PubMed Scopus (57) Google Scholar] (Fig. 1A). This is the reason why during inflammatory liver injury phagocyte-derived ROS primarily induce oncotic necrosis [2Jaeschke H. Reactive oxygen and mechanisms of inflammatory liver injury: present concepts.J Gastroenterol Hepatol. 2011; 26: 173-179Crossref PubMed Scopus (395) Google Scholar] (Fig. 1B and C). DisclosureThe underlying research reported in the study was funded by the NIH Institutes of Health. The underlying research reported in the study was funded by the NIH Institutes of Health.