Simulated Galactic Cosmic Rays Modify Mitochondrial Metabolism in Osteoclasts, Increase Osteoclastogenesis and Cause Trabecular Bone Loss in Mice.

Space is a high-stress environment. One major risk factor for the astronauts when they leave the Earth's magnetic field is exposure to ionizing radiation from galactic cosmic rays (GCR). Several adverse changes occur in mammalian anatomy and physiology in space, including bone loss. In this study, we assessed the effects of simplified GCR exposure on skeletal health in vivo. Three months following exposure to 0.5 Gy total body simulated GCR, blood, bone marrow and tissue were collected from 9 months old male mice. The key findings from our cell and tissue analysis are (1) GCR induced femoral trabecular bone loss in adult mice but had no effect on spinal trabecular bone. (2) GCR increased circulating osteoclast differentiation markers and osteoclast formation but did not alter new bone formation or osteoblast differentiation. (3) Steady-state levels of mitochondrial reactive oxygen species, mitochondrial and non-mitochondrial respiration were increased without any changes in mitochondrial mass in pre-osteoclasts after GCR exposure. (4) Alterations in substrate utilization following GCR exposure in pre-osteoclasts suggested a metabolic rewiring of mitochondria. Taken together, targeting radiation-mediated mitochondrial metabolic reprogramming of osteoclasts could be speculated as a viable therapeutic strategy for space travel induced bone loss.

TLR4-A Pertinent Player in Radiation-Induced Heart Disease?

The heart is one of the organs that is sensitive to developing delayed adverse effects of ionizing radiation (IR) exposure. Radiation-induced heart disease (RIHD) occurs in cancer patients and cancer survivors, as a side effect of radiation therapy of the chest, with manifestation several years post-radiotherapy. Moreover, the continued threat of nuclear bombs or terrorist attacks puts deployed military service members at risk of exposure to total or partial body irradiation. Individuals who survive acute injury from IR will experience delayed adverse effects that include fibrosis and chronic dysfunction of organ systems such as the heart within months to years after radiation exposure. Toll-like receptor 4 (TLR4) is an innate immune receptor that is implicated in several cardiovascular diseases. Studies in preclinical models have established the role of TLR4 as a driver of inflammation and associated cardiac fibrosis and dysfunction using transgenic models. This review explores the relevance of the TLR4 signaling pathway in radiation-induced inflammation and oxidative stress in acute as well as late effects on the heart tissue and the potential for the development of TLR4 inhibitors as a therapeutic target to treat or alleviate RIHD.

Effect of Sirt3 on hippocampal MnSOD activity, mitochondrial function, physiology, and cognition in an aged murine model.

The NAD(+)-dependent deacetylase SIRT3 is a proven mitochondrial metabolic stress sensor. It has been linked to the regulation of the mitochondrial acetylome and activation of several metabolic enzymes (e.g., manganese superoxide dismutase [MnSOD]) to protect mitochondrial function and redox homeostasis, which are vital for survival, excitability, and synaptic signaling of neurons mediating short- and long-term memory formation as well as retention. Eighteen-month-old male and female wild-type (WT) and Sirt3-/- mice were behaviorally tested for hippocampus-dependent cognitive performance in a Morris water maze paradigm. Cognitive impairment was displayed during the probe trial by female and male Sirt3-/- mice but not WT mice. Upon sacrifice, brains were fixed, and morphological assessments were conducted on hippocampal tissues. Both female and male Sirt3-/- mice demonstrated impaired spatial memory retention implying that SIRT3 plays a role in long-term memory function. Golgi-staining studies revealed decreased dendritic arborization and dendritic length in the hippocampi of male Sirt3-/- compared to WT animals. Sirt3 deletion significantly increased NR1, NR2A, and NR2B expression in the hippocampus of female mice only. Enzymatic activity of MnSOD, a major mitochondrial deacetylation target of SIRT3, was significantly decreased in both female and male Sirt3-/- mice. Similarly, both female and male Sirt3-/- mice demonstrated a significant decrease in their respiratory control ratio during Complex I-driven respiration, which was apparent only in female Sirt3-/- mice during Complex II-driven respiration.

Effects of Whole and Partial Heart Irradiation on Collagen, Mast Cells, and Toll-like Receptor 4 in the Mouse Heart.

In radiation therapy of tumors in the chest, such as in lung or esophageal cancer, part of the heart may be situated in the radiation field. This can lead to the development of radiation-induced heart disease. The mechanisms by which radiation causes long-term injury to the heart are not fully understood, but investigations in pre-clinical research models can contribute to mechanistic insights. Recent developments in X-ray technology have enabled partial heart irradiation in mouse models. In this study, adult male and female C57BL/6J mice were exposed to whole heart (a single dose of 8 or 16 Gy) and partial heart irradiation (16 Gy to 40% of the heart). Plasma samples were collected at 5 days and 2 weeks after the irradiation for metabolomics analysis, and the cardiac collagen deposition, mast cell numbers, and left ventricular expression of Toll-like receptor 4 (TLR4) were examined in the irradiated and unirradiated parts of the heart at 6 months after the irradiation. Small differences were found in the plasma metabolite profiles between the groups. However, the collagen deposition did not differ between the irradiated and unirradiated parts of the heart, and radiation did not upregulate the mast cell numbers in either part of the heart. Lastly, an increase in the expression of TLR4 was seen only after a single dose of 8 Gy to the whole heart. These results suggest that adverse tissue remodeling was not different between the irradiated and unirradiated portions of the mouse heart. While there were no clear differences between male and female animals, additional work in larger cohorts may be required to confirm this result, and to test the inhibition of TLR4 as an intervention strategy in radiation-induced heart disease.

Tocotrienols Provide Radioprotection to Multiple Organ Systems through Complementary Mechanisms of Antioxidant and Signaling Effects.

Tocotrienols have powerful radioprotective properties in multiple organ systems and are promising candidates for development as clinically effective radiation countermeasures. To facilitate their development as clinical radiation countermeasures, it is crucial to understand the mechanisms behind their powerful multi-organ radioprotective properties. In this context, their antioxidant effects are recognized for directly preventing oxidative damage to cellular biomolecules from ionizing radiation. However, there is a growing body of evidence indicating that the radioprotective mechanism of action for tocotrienols extends beyond their antioxidant properties. This raises a new pharmacological paradigm that tocotrienols are uniquely efficacious radioprotectors due to a synergistic combination of antioxidant and other signaling effects. In this review, we have covered the wide range of multi-organ radioprotective effects observed for tocotrienols and the mechanisms underlying it. These radioprotective effects for tocotrienols can be characterized as (1) direct cytoprotective effects, characteristic of the classic antioxidant properties, and (2) other effects that modulate a wide array of critical signaling factors involved in radiation injury.

A novel biotinylated lipid raft reporter for electron microscopic imaging of plasma membrane microdomains.

The submicroscopic spatial organization of cell surface receptors and plasma membrane signaling molecules is readily characterized by electron microscopy (EM) via immunogold labeling of plasma membrane sheets. Although various signaling molecules have been seen to segregate within plasma membrane microdomains, the biochemical identity of these microdomains and the factors affecting their formation are largely unknown. Lipid rafts are envisioned as submicron membrane subdomains of liquid ordered structure with differing lipid and protein constituents that define their specific varieties. To facilitate EM investigation of inner leaflet lipid rafts and the localization of membrane proteins therein, a unique genetically encoded reporter with the dually acylated raft-targeting motif of the Lck kinase was developed. This reporter, designated Lck-BAP-GFP, incorporates green fluorescent protein (GFP) and biotin acceptor peptide (BAP) modules, with the latter allowing its single-step labeling with streptavidin-gold. Lck-BAP-GFP was metabolically biotinylated in mammalian cells, distributed into low-density detergent-resistant membrane fractions, and was readily detected with avidin-based reagents. In EM images of plasma membrane sheets, the streptavidin-gold-labeled reporter was clustered in 20-50 nm microdomains, presumably representative of inner leaflet lipid rafts. The utility of the reporter was demonstrated in an investigation of the potential lipid raft localization of the epidermal growth factor receptor.

Small animal models of localized heart irradiation.

A subset of cancer patients treated with radiation therapy may experience radiation-induced heart disease (RIHD) that develops within weeks to several years after cancer treatment. Rodent models are most commonly used to examine the biological effects of local X-rays in the heart and test potential strategies to reduce RIHD. While developments in technology over the last decades have changed the procedures for local heart irradiation in animal models, the X-ray settings and radiation doses have remained quite consistent in time and between different research laboratories. This chapter provides a protocol for whole heart irradiation in rodent models, using an X-ray machine with cone beam computed tomography (CBCT) capabilities. Some methods for the quantification of common histological changes after whole heart irradiation in the rodent are also described.

Parthenolide induces rapid thiol oxidation that leads to ferroptosis in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is both a devastating and common disease. Every year in the United States, about 24,500 men and 10,000 women are diagnosed with HCC, and more than half of those diagnosed patients die from this disease. Thus far, conventional therapeutics have not been successful for patients with HCC due to various underlying comorbidities. Poor survival rate and high incidence of recurrence after therapy indicate that the differences between the redox environments of normal surrounding liver and HCC are valuable targets to improve treatment efficacy. Parthenolide (PTL) is a naturally found therapeutic with anti-cancer and anti-inflammatory properties. PTL can alter HCC's antioxidant environment through thiol modifications leaving tumor cells sensitive to elevated reactive oxygen species (ROS). Investigating the link between altered thiol mechanism and increased sensitivity to iron-mediated lipid peroxidation will allow for improved treatment of HCC. HepG2 (human) and McARH7777 (rat) HCC cells treated with PTL with increasing concentrations decrease cell viability and clonogenic efficiency in vitro. PTL increases glutathione (GSH) oxidation rescued by the addition of a GSH precursor, N-acetylcysteine (NAC). In addition, this elevation in thiol oxidation results in an overall increase in mitochondrial dysfunction. To elucidate if cell death is through lipid peroxidation, using a lipid peroxidation sensor indicated PTL increases lipid oxidation levels after 6 h. Additionally, western blotting reveals glutathione peroxidase 4 (GPx4) protein levels decrease after treatment with PTL suggesting cells are incapable of preventing lipid peroxidation after exposure to PTL. An elevation in lipid peroxidation will lead to a form of cell death known as ferroptosis. To further establish ferroptosis as a critical mechanism of death for HCC in vitro, the addition of ferrostatin-1 combined with PTL demonstrates a partial recovery in a colony survival assay. This study reveals that PTL can induce tumor cell death through elevations in intracellular oxidation, leaving cells sensitive to ferroptosis.

Transferrin receptor 1-mediated iron uptake regulates bone mass in mice via osteoclast mitochondria and cytoskeleton.

Increased intracellular iron spurs mitochondrial biogenesis and respiration to satisfy high-energy demand during osteoclast differentiation and bone-resorbing activities. Transferrin receptor 1 (Tfr1) mediates cellular iron uptake through endocytosis of iron-loaded transferrin, and its expression increases during osteoclast differentiation. Nonetheless, the precise functions of Tfr1 and Tfr1-mediated iron uptake in osteoclast biology and skeletal homeostasis remain incompletely understood. To investigate the role of Tfr1 in osteoclast lineage cells in vivo and in vitro, we crossed Tfrc (encoding Tfr1)-floxed mice with Lyz2 (LysM)-Cre and Cathepsin K (Ctsk)-Cre mice to generate Tfrc conditional knockout mice in myeloid osteoclast precursors (Tfr1ΔLysM) or differentiated osteoclasts (Tfr1ΔCtsk), respectively. Skeletal phenotyping by µCT and histology unveiled a significant increase in trabecular bone mass with normal osteoclast number in long bones of 10-week-old young and 6-month-old adult female but not male Tfr1ΔLysM mice. Although high trabecular bone volume in long bones was observed in both male and female Tfr1ΔCtsk mice, this phenotype was more pronounced in female knockout mice. Consistent with this gender-dependent phenomena, estrogen deficiency induced by ovariectomy decreased trabecular bone mass in Tfr1ΔLysM mice. Mechanistically, disruption of Tfr1 expression attenuated mitochondrial metabolism and cytoskeletal organization in mature osteoclasts in vitro by attenuating mitochondrial respiration and activation of the Src-Rac1-WAVE regulatory complex axis, respectively, leading to decreased bone resorption with little impact on osteoclast differentiation. These results indicate that Tfr1-mediated iron uptake is specifically required for osteoclast function and is indispensable for bone remodeling in a gender-dependent manner.

Sex-dependent effects of genetic upregulation of activated protein C on delayed effects of acute radiation exposure in the mouse heart, small intestine, and skin.

Accidental exposure to ionizing radiation may lead to delayed effects of acute radiation exposure (DEARE) in many organ systems. Activated protein C (APC) is a known mitigator of the acute radiation syndrome. To examine the role of APC in DEARE, we used a transgenic mouse model with 2- to 3-fold increased plasma levels of APC (high in APC, APCHi). Male and female APCHi mice and wild-type littermates were exposed to 9.5 Gy γ-rays with their hind-legs (bone marrow) shielded from radiation to allow long-term survival. At 3 and 6 months after irradiation, cardiac function was measured with ultrasonography. At 3 months, radiation increased cardiac dimensions in APCHi males, while decreases were seen in wild-type females. At this early time point, APCHi mice of both sexes were more susceptible to radiation-induced changes in systolic function compared to wild-types. At 6 months, a decrease in systolic function was mainly seen in male mice of both genotypes. At 6 months, specimens of heart, small intestine and dorsal skin were collected for tissue analysis. Female APCHi mice showed the most severe radiation-induced deposition of cardiac collagens but were protected against a radiation-induced loss of microvascular density. Both male and female APCHi mice were protected against a radiation induced upregulation of toll-like receptor 4 in the heart, but this did not translate into a clear protection against immune cell infiltration. In the small intestine, the APCHi genotype had no effect on an increase in the number of myeloperoxidase positive cells (seen mostly in females) or an increase in the expression of T-cell marker CD2 (males). Lastly, both male and female APCHi mice were protected against radiation-induced epidermal thickening and increase in 3-nitrotyrosine positive keratinocytes. In conclusion, prolonged high levels of APC in a transgenic mouse model had little effects on indicators of DEARE in the heart, small intestine and skin, with some differential effects in male compared to female mice.

Assessment of Cellular Oxidation using a Subcellular Compartment-Specific Redox-Sensitive Green Fluorescent Protein.

Measuring the intracellular oxidation/reduction balance provides an overview of the physiological and/or pathophysiological redox status of an organism. Thiols are especially important for illuminating the redox status of cells via their reduced dithiol and oxidized disulfide ratios. Engineered cysteine-containing fluorescent proteins open a new era for redox-sensitive biosensors. One of them, redox-sensitive green fluorescent protein (roGFP), can easily be introduced into cells with adenoviral transduction, allowing the redox status of subcellular compartments to be evaluated without disrupting cellular processes. Reduced cysteines and oxidized cystines of roGFP have excitation maxima at 488 nm and 405 nm, respectively, with emission at 525 nm. Assessing the ratios of these reduced and oxidized forms allows the convenient calculation of redox balance within the cell. In this method article, immortalized human triple-negative breast cancer cells (MDA-MB-231) were used to assess redox status within the living cell. The protocol steps include MDA-MB-231 cell line transduction with adenovirus to express cytosolic roGFP, treatment with H2O2, and assessment of cysteine and cystine ratio with both flow cytometry and fluorescence microscopy.

Fractionated radiation suppresses Kruppel-like factor 2 pathway to a greater extent than by single exposure to the same total dose.

Kruppel-like factor 2 (KLF2) is a positive transcriptional regulator of several endothelial protective molecules, including thrombomodulin (TM), a surface receptor, and endothelial nitric oxide synthase (eNOS), an enzyme that generates nitric oxide (NO). Loss of TM and eNOS causes endothelial dysfunction, which results in suppressed generation of activated protein C (APC) by TM-thrombin complex and in upregulation of intercellular adhesion molecule 1 (ICAM-1). Mechanistic studies revealed that activation of extracellular signal-regulated kinase 5 (ERK5) via upregulation of myocyte enhancer factor 2 (MEF2) induces KLF2 expression. Radiation causes endothelial dysfunction, but no study has investigated radiation's effects on the KLF2 pathway. Because fractionated radiation is routinely used during cancer radiotherapy, we decided to delineate the effects of radiation dose fractionation on the KLF2 signaling cascade at early time points (up to 24 h). We exposed human primary endothelial cells to radiation as a series of fractionated or as a single exposure, with the same total dose delivered to each group. We measured the expression and activity of critical members of the KLF2 pathway at subsequent time points, and determined whether pharmacological upregulation of KLF2 can reverse the radiation effects. Compared to single exposure, fractionated radiation profoundly suppressed KLF2, TM, and eNOS levels, subdued APC generation, declined KLF2 binding ability to TM and eNOS promoters, enhanced ICAM-1 expression, and decreased expression of upstream regulators of KLF2 (ERK5 and MEF2). Pharmacological inhibitors of the mevalonate pathway prevented fractionated-radiation-induced suppression of KLF2, TM, and eNOS expression. Finally, fractionated irradiation to thoracic region more profoundly suppressed KLF2 and enhanced ICAM-1 expression than single exposure in the lung at 24 h. These data clearly indicate that radiation dose fractionation plays a critical role in modulating levels of KLF2, its upstream regulators, and its downstream target molecules in endothelial cells. Our findings will provide important insights for selecting fractionated regimens during radiotherapy and for developing strategies to alleviate radiotherapy-induced toxicity to healthy tissues.

The Role of Sirtuin 3 in Radiation-Induced Long-Term Persistent Liver Injury.

In patients with abdominal region cancers, ionizing radiation (IR)-induced long-term liver injury is a major limiting factor in the use of radiotherapy. Previously, the major mitochondrial deacetylase, sirtuin 3 (SIRT3), has been implicated to play an important role in the development of acute liver injury after total body irradiation but no studies to date have examined the role of SIRT3 in liver's chronic response to radiation. In the current study, ten-month-old Sirt3-/- and Sirt3+/+ male mice received 24 Gy radiation targeted to liver. Six months after exposure, irradiated Sirt3-/- mice livers demonstrated histopathological elevations in inflammatory infiltration, the loss of mature bile ducts and higher DNA damage (TUNEL) as well as protein oxidation (3-nitrotyrosine). In addition, increased expression of inflammatory chemokines (IL-6, IL-1ß, TGF-ß) and fibrotic factors (Procollagen 1, α-SMA) were also measured in Sirt3-/- mice following 24 Gy IR. The alterations measured in enzymatic activities of catalase, glutathione peroxidase, and glutathione reductase in the livers of irradiated Sirt3-/- mice also implied that hydrogen peroxide and hydroperoxide sensitive signaling cascades in the absence of SIRT3 might contribute to the IR-induced long-term liver injury.

Epigenetic Control of Cdkn2a.Arf Protects Tumor-Infiltrating Lymphocytes from Metabolic Exhaustion.

T-cell exhaustion in cancer is linked to poor clinical outcomes, where evidence suggests T-cell metabolic changes precede functional exhaustion. Direct competition between tumor-infiltrating lymphocytes (TIL) and cancer cells for metabolic resources often renders T cells dysfunctional. Environmental stress produces epigenome remodeling events within TIL resulting from loss of the histone methyltransferase EZH2. Here, we report an epigenetic mechanism contributing to the development of metabolic exhaustion in TIL. A multiomics approach revealed a Cdkn2a.Arf-mediated, p53-independent mechanism by which EZH2 inhibition leads to mitochondrial dysfunction and the resultant exhaustion. Reprogramming T cells to express a gain-of-function EZH2 mutant resulted in an enhanced ability of T cells to inhibit tumor growth in vitro and in vivo. Our data suggest that manipulation of T-cell EZH2 within the context of cellular therapies may yield lymphocytes that are able to withstand harsh tumor metabolic environments and collateral pharmacologic insults. SIGNIFICANCE: These findings demonstrate that manipulation of T-cell EZH2 in cellular therapies may yield cellular products able to withstand solid tumor metabolic-deficient environments. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/21/4707/F1.large.jpg.

Antioxidant Tocols as Radiation Countermeasures (Challenges to be Addressed to Use Tocols as Radiation Countermeasures in Humans).

Radiation countermeasures fall under three categories, radiation protectors, radiation mitigators, and radiation therapeutics. Radiation protectors are agents that are administered before radiation exposure to protect from radiation-induced injuries by numerous mechanisms, including scavenging free radicals that are generated by initial radiochemical events. Radiation mitigators are agents that are administered after the exposure of radiation but before the onset of symptoms by accelerating the recovery and repair from radiation-induced injuries. Whereas radiation therapeutic agents administered after the onset of symptoms act by regenerating the tissues that are injured by radiation. Vitamin E is an antioxidant that neutralizes free radicals generated by radiation exposure by donating H atoms. The vitamin E family consists of eight different vitamers, including four tocopherols and four tocotrienols. Though alpha-tocopherol was extensively studied in the past, tocotrienols have recently gained attention as radiation countermeasures. Despite several studies performed on tocotrienols, there is no clear evidence on the factors that are responsible for their superior radiation protection properties over tocopherols. Their absorption and bioavailability are also not well understood. In this review, we discuss tocopherol's and tocotrienol's efficacy as radiation countermeasures and identify the challenges to be addressed to develop them into radiation countermeasures for human use in the event of radiological emergencies.

PCB11 Metabolite, 3,3'-Dichlorobiphenyl-4-ol, Exposure Alters the Expression of Genes Governing Fatty Acid Metabolism in the Absence of Functional Sirtuin 3: Examining the Contribution of MnSOD.

Although the production of polychlorinated biphenyls (PCBs) is prohibited, the inadvertent production of certain lower-chlorinated PCB congeners still threatens human health. We and others have identified 3,3'-dichlorobiphenyl (PCB11) and its metabolite, 3,3'-dichlorobiphenyl-4-ol (4OH-PCB11), in human blood, and there is a correlation between exposure to this metabolite and mitochondrial oxidative stress in mammalian cells. Here, we evaluated the downstream effects of 4OH-PCB11 on mitochondrial metabolism and function in the presence and absence of functional Sirtuin 3 (SIRT3), a mitochondrial fidelity protein that protects redox homeostasis. A 24 h exposure to 3 µM 4OH-PCB11 significantly decreased the cellular growth and mitochondrial membrane potential of SIRT3-knockout mouse embryonic fibroblasts (MEFs). Only wild-type cells demonstrated an increase in Manganese superoxide dismutase (MnSOD) activity in response to 4OH-PCB11⁻induced oxidative injury. This suggests the presence of a SIRT3-mediated post-translational modification to MnSOD, which was impaired in SIRT3-knockout MEFs, which counters the PCB insult. We found that 4OH-PCB11 increased mitochondrial respiration and endogenous fatty-acid oxidation-associated oxygen consumption in SIRT3-knockout MEFs; this appeared to occur because the cells exhausted their reserve respiratory capacity. To determine whether these changes in mitochondrial respiration were accompanied by similar changes in the regulation of fatty acid metabolism, we performed quantitative real-time polymerase chain reaction (qRT-PCR) after a 24 h treatment with 4OH-PCB11. In SIRT3-knockout MEFs, 4OH-PCB11 significantly increased the expression of ten genes controlling fatty acid biosynthesis, metabolism, and transport. When we overexpressed MnSOD in these cells, the expression of six of these genes returned to the baseline level, suggesting that the protective role of SIRT3 against 4OH-PCB11 is partially governed by MnSOD activity.

Trifluoperazine inhibits acetaminophen-induced hepatotoxicity and hepatic reactive nitrogen formation in mice and in freshly isolated hepatocytes.

The hepatotoxicity of acetaminophen (APAP) occurs by initial metabolism to N-acetyl-p-benzoquinone imine which depletes GSH and forms APAP-protein adducts. Subsequently, the reactive nitrogen species peroxynitrite is formed from nitric oxide (NO) and superoxide leading to 3-nitrotyrosine in proteins. Toxicity occurs with inhibited mitochondrial function. We previously reported that in hepatocytes the nNOS (NOS1) inhibitor NANT inhibited APAP toxicity, reactive nitrogen and oxygen species formation, and mitochondrial dysfunction. In this work we examined the effect of trifluoperazine (TFP), a calmodulin antagonist that inhibits calcium induced nNOS activation, on APAP hepatotoxicity and reactive nitrogen formation in murine hepatocytes and in vivo. In freshly isolated hepatocytes TFP inhibited APAP induced toxicity, reactive nitrogen formation (NO, GSNO, and 3-nitrotyrosine in protein), reactive oxygen formation (superoxide), loss of mitochondrial membrane potential, decreased ATP production, decreased oxygen consumption rate, and increased NADH accumulation. TFP did not alter APAP induced GSH depletion in the hepatocytes or the formation of APAP protein adducts which indicated that reactive metabolite formation was not inhibited. Since we previously reported that TFP inhibits the hepatotoxicity of APAP in mice without altering hepatic APAP-protein adduct formation, we examined the APAP treated mouse livers for evidence of reactive nitrogen formation. 3-Nitrotyrosine in hepatic proteins and GSNO were significantly increased in APAP treated mouse livers and decreased in the livers of mice treated with APAP plus TFP. These data are consistent with a hypothesis that APAP hepatotoxicity occurs with altered calcium metabolism, activation of nNOS leading to increased reactive nitrogen formation, and mitochondrial dysfunction.

Metabolically Biotinylated Reporters for Electron Microscopic Imaging of Cytoplasmic Membrane Microdomains.

The protein and lipid substituents of cytoplasmic membranes are not in general homogeneously distributed across the membrane surface. Many membrane proteins, including ion channels, receptors, and other signaling molecules, exhibit a profound submicroscopic spatial organization, in some cases clustering in submicron membrane subdomains having a protein and lipid composition distinct from that of the bulk membrane. In the case of membrane-associated signaling molecules, mounting evidence indicates that their nanoscale organization, for example the colocalization of differing signaling molecules in the same membrane microdomains versus their segregation into distinct microdomain species, can significantly impact signal transduction. Biochemical membrane fractionation approaches have been used to characterize membrane subdomains of unique protein and lipid composition, including cholesterol-rich lipid raft structures. However, the intrinsically perturbing nature of fractionation methods makes the interpretation of such characterization subject to question, and indeed the existence and significance of lipid rafts remain controversial. Electron microscopic (EM) imaging of immunogold-labeled proteins in plasma membrane sheets has emerged as a powerful method for visualizing the nanoscale organization and colocalization of membrane proteins, which is not as perturbing of membrane structure as are biochemical approaches. For the purpose of imaging putative lipid raft structures, we recently developed a streamlined EM membrane sheet imaging procedure that employs a unique genetically encoded and metabolically biotinylated reporter that is targeted to membrane inner leaflet lipid rafts. We describe here the principles of this procedure and its application in the imaging of plasma membrane inner leaflet lipid rafts.

Loss of C/EBPδ enhances IR-induced cell death by promoting oxidative stress and mitochondrial dysfunction.

Exposure of cells to ionizing radiation (IR) generates reactive oxygen species (ROS). This results in increased oxidative stress and DNA double strand breaks (DSBs) which are the two underlying mechanisms by which IR causes cell/tissue injury. Cells that are deficient or impaired in the cellular antioxidant response are susceptible to IR-induced apoptosis. The transcription factor CCAAT enhancer binding protein delta (Cebpd, C/EBPδ) has been implicated in the regulation of oxidative stress, DNA damage response, genomic stability and inflammation. We previously reported that Cebpd-deficient mice are sensitive to IR and display intestinal and hematopoietic injury, however the underlying mechanism is not known. In this study, we investigated whether an impaired ability to detoxify IR-induced ROS was the underlying cause of the increased radiosensitivity of Cebpd-deficient cells. We found that Cebpd-knockout (KO) mouse embryonic fibroblasts (MEFs) expressed elevated levels of ROS, both at basal levels and after exposure to gamma radiation which correlated with increased apoptosis, and decreased clonogenic survival. Pre-treatment of wild type (WT) and KO MEFs with polyethylene glycol-conjugated Cu-Zn superoxide dismutase (PEG-SOD) and catalase (PEG-CAT) combination prior to irradiation showed a partial rescue of clonogenic survival, thus demonstrating a role for increased intracellular oxidants in promoting IR-induced cell death. Analysis of mitochondrial bioenergetics revealed that irradiated KO MEFs showed significant reductions in basal, adenosine triphosphate (ATP)-linked, maximal respiration and reserved respiratory capacity and decrease in intracellular ATP levels compared to WT MEFs indicating they display mitochondrial dysfunction. KO MEFs expressed significantly lower levels of the cellular antioxidant glutathione (GSH) and its precursor- cysteine as well as methionine. In addition to its antioxidant function, GSH plays an important role in detoxification of lipid peroxidation products such as 4-hydroxynonenal (4-HNE). The reduced GSH levels observed in KO MEFs correlated with elevated levels of 4-HNE protein adducts in irradiated KO MEFs compared to respective WT MEFs. We further showed that pre-treatment with the GSH precursor, N-acetyl L-cysteine (NAC) prior to irradiation showed a significant reduction of IR-induced cell death and increases in GSH levels, which contributed to the overall increase in clonogenic survival of KO MEFs. In contrast, pre-treatment with the GSH synthesis inhibitor- buthionine sulfoximine (BSO) further reduced the clonogenic survival of irradiated KO MEFs. This study demonstrates a novel role for C/EBPδ in protection from basal as well as IR-induced oxidative stress and mitochondrial dysfunction thus promoting post-radiation survival.

The neuronal nitric oxide synthase inhibitor NANT blocks acetaminophen toxicity and protein nitration in freshly isolated hepatocytes.

3-Nitrotyrosine (3NT) in liver proteins of mice treated with hepatotoxic doses of acetaminophen (APAP) has been postulated to be causative in toxicity. Nitration is by a reactive nitrogen species formed from nitric oxide (NO). The source of the NO is unclear. iNOS knockout mice were previously found to be equally susceptible to APAP toxicity as wildtype mice and iNOS inhibitors did not decrease toxicity in mice or in hepatocytes. In this work we examined the potential role of nNOS in APAP toxicity in hepatocytes using the specific nNOS inhibitor NANT (10 µM)(N-[(4S)-4-amino-5-[(2-aminoethyl)amino]pentyl]-N'-nitroguanidinetris (trifluoroacetate)). Primary hepatocytes (1 million/ml) from male B6C3F1 mice were incubated with APAP (1mM). Cells were removed and assayed spectrofluorometrically for reactive nitrogen and oxygen species using diaminofluorescein (DAF) and Mitosox red, respectively. Cytotoxicity was determined by LDH release into media. Glutathione (GSH, GSSG), 3NT, GSNO, acetaminophen-cysteine adducts, NAD, and NADH were measured by HPLC. APAP significantly increased cytotoxicity at 1.5-3.0 h. The increase was blocked by NANT. NANT did not alter APAP mediated GSH depletion or acetaminophen-cysteine adducts in proteins which indicated that NANT did not inhibit metabolism. APAP significantly increased spectroflurometric evidence of reactive nitrogen and oxygen formation at 0.5 and 1.0 h, respectively, and increased 3NT and GSNO at 1.5-3.0 h. These increases were blocked by NANT. APAP dramatically increased NADH from 0.5-3.0 h and this increase was blocked by NANT. Also, APAP decreased the Oxygen Consumption Rate (OCR), decreased ATP production, and caused a loss of mitochondrial membrane potential, which were all blocked by NANT.