Sleep Deprivation Death Linked Causally to the Gut

Reporter : Irina Robu, PhD

Neuroscientists at Harvard Medical School identified an unexpected link between sleep deprivation and premature death. Their findings show that the possibility that animals might be able to survive without sleep, under certain circumstances. Their study with sleep-deprived fruit flies found that death was continuously by the accumulation of reactive oxidative species in the gut. And when the flies were given antioxidant compounds that neutralized and cleared ROS from the gut, the sleep-deprived animals remained active and had normal lifespans. Extra experiments in mice confirmed that ROS accumulated in the gut when they didn’t get enough sleep.

Yet, in spite of decades of study, researchers still haven’t revealed why animals die when they don’t sleep. In attempts to answer how sleep deprivation culminates in death, most research has focused on the brain, where sleep originates. However, studies have yet to yield conclusive results. In addition to impairing cognition, sleep loss leads to dysfunction of the gastrointestinal, immune, metabolic, and circulatory systems.

The Harvard Medical School team carried out a sequence of experiments in fruit flies to search throughout the body for signs of damage caused by sleep deprivation. Fruit flies share many sleep-regulating genes with humans. To screen sleep, the investigators used infrared beams to constantly track the movement of flies housed in individual tubes. Scientist show that flies can sleep through physical shaking, so they genetically manipulated fruit flies to express a heat-sensitive protein in specific neurons, the activity of which are known to suppress sleep. When flies were housed at 29°C the protein induced neurons to remain constantly active, thus preventing the flies from sleeping.

The scientists discovered that fruit fly mortality spiked after 10 days of temperature-induced sleep deprivation and all of the flies died by around day 20 and control flies that had normal sleep lived up to approximately 40 days in the same environmental conditions. Since mortality increased around day 10, the scientists looked for markers of cell damage on that and the preceding days. They noticed that the guts of sleep-deprived flies had a dramatic build-up of ROS. The buildup of ROS in the fruit fly guts peaked around day 10 of sleep deprivation, and when deprivation was stopped, ROS levels decreased.

To find out if ROS in the gut plays a causal role in sleep deprivation-induced death, the researchers next looked at whether preventing ROS accumulation could prolong survival. They tested dozens of compounds with antioxidant properties known to neutralize ROS and identified 11 that, when given as a food supplement, allowed sleep-deprived flies to have a normal or near-normal lifespan. These compounds, such as melatonin, lipoic acid, and NAD, were particularly effective at clearing ROS from the gut. Notably, the supplements did not extend the lifespan of non-sleep-deprived flies.

The role of ROS removal in preventing death was also confirmed by experiments in which flies were genetically manipulated to overproduce antioxidant enzymes in their guts. These flies had normal to near-normal lifespans when sleep deprived, but flies that overproduced antioxidant enzymes in their nervous systems weren’t protected from sleep-deprivation-related death. While the results demonstrated that ROS build up in the gut plays a central role in causing premature death from sleep deprivation, the researchers acknowledged that many questions are still without answers. At the same time, they found that insufficient sleep is identified to restrict with the body’s hunger signaling pathways, which lead to measure the fruit fly food intake to analyze whether there were potential associations between feeding and death. They found that some sleep-deprived flies ate more throughout the day compared with non-deprived controls.

The researchers are now working to identify the biological pathways that lead to ROS accumulation in the gut and subsequent physiological disruptions.

SOURCE

Death Due to Sleep Deprivation Linked Causally to the Gut, and is Preventable in Flies

 

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Metabolic insight into cancer cell survival

Metabolic insight into cancer cell survival

Larry H. Bernstein, MD, FCAP, Curator

LPBI

UPDATED 9/26/2021

Periprostatic Adipose Tissue Favors Prostate Cancer Cell Invasion in an Obesity-Dependent Manner: Role of Oxidative Stress

Victor Laurent, Aurélie Toulet, Camille Attané, Delphine Milhas, Stéphanie Dauvillier, Falek Zaidi, Emily Clement, Mathieu Cinato, Sophie Le Gonidec, Adrien Guérard, Camille Lehuédé, David Garandeau, Laurence Nieto, Edith Renaud-Gabardos, Anne-Catherine Prats, Philippe Valet, Bernard Malavaud and Catherine Muller

Source: https://mcr.aacrjournals.org/content/17/3/821

Abstract

Prostate gland is surrounded by periprostatic adipose tissue (PPAT), which is increasingly believed to play a paracrine role in prostate cancer progression. Our previous work demonstrates that adipocytes promote homing of prostate cancer cells to PPAT and that this effect is upregulated by obesity. Here, we show that once tumor cells have invaded PPAT (mimicked by an in vitro model of coculture), they establish a bidirectional crosstalk with adipocytes, which promotes tumor cell invasion. Indeed, tumor cells induce adipocyte lipolysis and the free fatty acids (FFA) released are taken up and stored by tumor cells. Incubation with exogenous lipids also stimulates tumor cell invasion, underlining the importance of lipid transfer in prostate cancer aggressiveness. Transferred FFAs (after coculture or exogenous lipid treatment) stimulate the expression of one isoform of the pro-oxidant enzyme NADPH oxidase, NOX5. NOX5 increases intracellular reactive oxygen species (ROS) that, in turn, activate a HIF1/MMP14 pathway, which is responsible for the increased tumor cell invasion. In obesity, tumor-surrounding adipocytes are more prone to activate the depicted signaling pathway and to induce tumor invasion. Finally, the expression of NOX5 and MMP14 is upregulated at the invasive front of human tumors where cancer cells are in close proximity to adipocytes and this process is amplified in obese patients, underlining the clinical relevance of our results.

Implications: Our work emphasizes the key role of adjacent PPAT in prostate cancer dissemination and proposes new molecular targets for the treatment of obese patients exhibiting aggressive diseases.

Introduction

Malignant evolution of solid cancers relies on complex cell-to-cell interactions sustained by a broad network of physical and chemical mediators that constitute the tumor microenvironment (1). Adipose tissue, and its main cellular component, adipocytes, have recently emerged as key actors in solid tumor progression (2). Upon proximal adipose tissue infiltration, a bidirectional crosstalk takes place between invasive tumor cells and adipocytes. Initially described in breast cancer, tumor-surrounding adipocytes exhibit extensive phenotypical changes defined by a decrease in lipid content, a decreased expression of adipocyte markers, and an activated state demonstrated predominantly by the overexpression of proinflammatory cytokines and ECM (extracellular matrix)-related molecules (3). We named these cells cancer-associated adipocytes (CAA; ref. 3). Occurrence of CAAs is not restricted to breast cancer and has been described in a wide range of solid tumors including metastatic ovarian cancer, renal and colon cancers, as well as melanoma (2). In turn, CAAs promote tumor aggressiveness by secreting soluble factors, ECM proteins, and ECM-remodeling enzymes and by modulating tumor cell metabolism (2). The decrease in size and lipid content of tumor-surrounding adipocytes results from a “dedifferentiation” process depending on the reactivation of the Wnt/β-catenin pathway in response to Wnt3a secreted by tumor cells (4) and from induction of lipolysis, the latter process resulting in release of free fatty acids (FFA; refs. 5–7). These FFAs are then taken up, stored in lipid droplets, and used by tumor cells, where they have been described to contribute to tumor progression mainly through modulation of tumor cell metabolism toward fatty acid oxidation (FAO; refs. 5–7). This metabolic symbiosis instilled between cancer cells and tumor-surrounding adipocytes is only beginning to be explored but could provide new therapeutic targets as we recently reviewed in ref. 8. In addition to FAO, FFAs acquired from adipocytes could be used as membrane building blocks and/or signaling lipids and the fate of these lipids may depend on the tumor cell type.

Among the different types of tumors whose close interaction with adipose tissue could influence tumor progression is prostate cancer, the most common malignancy in men in Western countries. The prostate is surrounded by periprostatic adipose tissue (PPAT) and extraprostatic extension into PPAT is a widely acknowledged adverse prognostic factor in prostate cancer and an important determinant of prostate cancer recurrence after treatment (9). The positive association between obesity and aggressive prostate cancer, defined by an increase in local and distant dissemination, is also in favor of a role for adipose tissue in tumor progression (10). We have recently demonstrated that adipocytes from PPAT favor the initial step of PPAT infiltration by secreting the CCL7 chemokine that attracts CCR3-expressing cancer cells, and this process is amplified in obesity (11). Prostate-confined cancer cell migration and invasion may also be promoted at distance by inflammatory cytokines and metalloproteases secreted by PPAT, as well as by adipocyte-derived exosomes (12–14). In contrast to other cancer types such as breast or ovarian cancer (2), the effect of the invaded cancer cells on adipocytes within PPAT has been poorly described. Secretions from PPAT are modified by tumor-conditioned medium with upregulation of osteopontin, TNFα, and IL6, highlighting that, like other adipose depots, it is not inert to tumors (15). Coculture of prostate cancer cells with rat epididymal adipocytes increases growth and changes the morphology of prostate cancer cells (16, 17), but phenotypical changes of adipocytes have not been investigated in these studies. In addition, the mechanisms that govern increased prostate cancer cell aggressiveness in the presence of adipocytes are poorly described and have been mainly attributed to soluble factors (such as IL6; for review, see ref. 18). Finally, lipid transfer has not been demonstrated between periprostatic adipocytes and tumor cells, but only with bone marrow–derived adipocytes present at prostate cancer’s most frequent metastatic site (19, 20). Here, we demonstrate that prostate cancer cells are able to induce a CAA phenotype in vitro and in vivo, and that CAAs, in turn, promote tumor invasion. Lipid transfer between tumor-surrounding adipocytes and cancer cells promotes tumor aggressiveness by inducing oxidative stress in a NADPH oxidase–dependent manner, activating a proinvasive signaling pathway. The overall pathway is amplified in obesity and has been validated in human tumors. This study highlights the importance of lipid transfer in the tumor-promoting effect of adipocytes but also underlines that the consequence of this process is not univocal among all tumor types.

Revised 4/20/2016

AACR 2016: Novel Epigenetic Drug Therapeutics Revealed

http://www.genengnews.com/gen-news-highlights/aacr-2016-epigenetic-drug-therapeutics/81252634/

As the 2016 American Association for Cancer Research meeting begins to downshift toward a close, the presentation sessions certainly did not suffer from a lack of enthusiasm from attendees or high-quality research from presenters. Of particular note was a major symposium that discussed next-generation epigenetic therapeutics.

In the past several years, there have been a variety of epigenetic targets exploited by newly developed drug compounds, many of which have already progressed into clinical trials. Often these compounds will target specific classes of epigenetic regulators like acetylases and histone demethylases, for instance, the small-molecule inhibitors of protein interacting bromodomains—implicated in a diverse range of cancers—and methyltransferase inhibitors, such as lysyl demethylases (KDM).

However, for all of their recently achieved success, researchers are continually searching for increasingly rapid methods to validate epigenetic drug targets. Session Chairperson Udo Oppermann, Ph.D., principal investigator at the University of Oxford, stressed that open access research and continued investigator cooperation were key factors for driving rapid development of novel therapeutics in the field. Anecdotally, Dr. Oppermann noted that if biologists were a bit more like the international cooperative teams of physicists that discovered the Higgs boson or gravitational waves, many biological endeavors would advance rather quickly.

After providing the audience with a brief introduction to the symposium’s topic, Dr. Oppermann described his current research on histone demethylase inhibitors in multiple myeloma and the connection to metabolic pathways. He surmised that tricarboxylic acid (TCA) cycle-derived metabolites can link cellular metabolism to cancer—impacting epigenetic landscapes. Specifically, the TCA intermediates are inhibitors of KDMs, ultimately controlling epigenetic and metabolic regulation.

Furthermore, Dr. Oppermann’s group was able to show that treatment of myeloma cell lines with the potent and specific histone demethylase inhibitor GSK-J4 was able to reverse the Myc-driven metabolic dependency, forcing a selected amino acid depletion. This deficiency led to the integrated stress response and the activation of proapoptotic genes. This work helps to solidify further the potent nature of GSK-J4 in cancer while simultaneously uncovering the metabolic mechanisms that cancer cells employ to keep their overproliferative phenotypes progressing forward.

Next, Tomasz Cierpicki, Ph.D., assistant professor at the University of Michigan, described his work on targeting leukemic stem cells with small-molecule inhibitors of the protein regulator of cytokinesis 1 (PRC1). Dr. Cierpicki took the audience through his research design, which was to target BMI1, an oncogene that determines the proliferative capacity and self-renewal potential of normal and leukemic stem cells. BMI1 has been implicated in a variety of tumors and is essential for the Polycomb Repressive Complex 1 (PRC1). Moreover, BMI1 interacts with the RING1B protein to form an active E3 ubiquitin ligase that targets histone H2A, modifying epigenetic regulation mechanisms.

Dr. Cierpicki’s laboratory looked at inhibitors of the RING1B–BMI1 E3 ligase complex as potential therapeutic agents targeting cancer stem cells. Using an array of techniques from fragment screening to medicinal chemistry, the researchers were able to identify potent compounds that bound to RING1B–BMI1 and inhibit its E3 ubiquitin ligase activity with low micromolar affinities. When testing in vitro, the inhibitors revealed robust downregulation of H2A ubiquitination. Dr. Cierpicki and his colleagues found that the RING1–BMI1 inhibitor blocked the self-renewal capacity of the stem cells and induced cellular differentiation—validating RING ligases as a novel epigenetic drug target.

Finishing up the session was William Sellers, M.D., vice president and global head of oncology for the Novartis Institutes for BioMedical Research. Dr. Sellers’ research is focused on what genes are necessary for epigenetic regulation of cancer and how they are linked to essential metabolic processes. He and his colleagues accomplished their studies through the use of large-scale shRNA screening across a diverse set of 390 cancer cell lines.

Utilizing deep small hairpin RNA (shRNA) screening libraries, at 20 shRNAs per genome, provided the investigators with highly robust gene-level data, which resulted in the emergence of several distinct classes of cancer-dependent genes. For example, Dr. Sellers’ group found that several known oncogenes fell into the genetic dependence class, whereas other genes were sorted into lineage, paralog, and collateral synthetic lethality dependent classes.

An interesting example from Dr. Sellers’ work was the link his laboratory discovered between polyamine metabolism and salvage and the protein arginine methyltransferase 5 (PRMT5). In particular, the loss of methylthioadenosine phosphorylase (MTAP)—which has been observed in many solid tumors and hematologic malignancies—resulted in the accumulation of S-methyl-5′-thioadenosine (MTA), which specifically inhibited the epigenetic mechanisms of PRMT5. The culmination Dr. Sellers’ analysis led to the finding that PRMT5 is a novel target for therapeutic development in MTAP deleted cancers.

These three presentations represented some of the excellent, cutting-edge research that is not only looking to develop novel drug therapeutics but also trying to uncover the underlying molecular mechanisms of epigenetic regulation and cancer.

A Metabolic Twist that Drives Cancer Survival

http://www.technologynetworks.com/Metabolomics/news.aspx?ID=190262

A novel metabolic pathway that helps cancer cells thrive in conditions that are lethal to normal cells has been identified.

“It’s long been thought that if we could target tumor-specific metabolic pathways, it could lead to effective ways to treat cancer,” said senior author Dr. Ralph DeBerardinis, Associate Professor of CRI and Pediatrics, Director of CRI’s Genetic and Metabolic Disease Program, and Chief of the Division of Pediatric Genetics and Metabolism at UT Southwestern. “This study finds that two very different metabolic processes are linked in a way that is specifically required for cells to adapt to the stress associated with cancer progression.”

The study, available online today in the journal Nature, reveals that cancer cells use an alternate version of two well-known metabolic pathways called the pentose phosphate pathway (PPP) and the Krebs cycle to defend against toxins. The toxins are reactive oxygen species (ROS) that kill cells via oxidative stress.

This work builds on earlier studies by Dr. DeBerardinis’ laboratory that found the Krebs cycle, a series of chemical reactions that cells use to generate energy, could reverse itself under certain conditions to nourish cancer cells.

Dr. DeBerardinis said most normal cells and tumor cells grow by attaching to nutrient-rich tissue called a matrix. “They are dependent on matrix attachment to receive growth-promoting signals and to regulate their metabolism in a way that supports cell growth, proliferation, and survival,” he said.

Detachment from the matrix results in a sudden increase in ROS that is lethal to normal cells, he added. Cancer cells seem to have a workaround.

The destruction of healthy cells when detached from the matrix was reported in a landmark 2009 Nature study by Harvard Medical School cell biologist Dr. Joan Brugge. Intriguingly, that same study found that inserting an oncogene – a gene with the potential to cause cancer – into a normal cell caused it to behave like a cancer cell and survive detachment, said Dr. DeBerardinis, who also is affiliated with the Eugene McDermott Center for Human Growth & Development, holds the Joel B. Steinberg, M.D. Chair in Pediatrics, and is a Sowell Family Scholar in Medical Research at UT Southwestern.

“Another Nature study, this one from CRI Director Dr. Sean Morrison’s laboratory in November 2015, found that the rare skin cancer cells that were able to detach from the primary tumor and successfully metastasize to other parts of the body had the ability to keep ROS levels from getting dangerously high,” Dr. DeBerardinis said. Dr. Morrison, also a CPRIT Scholar in Cancer Research and a Howard Hughes Medical Institute Investigator, holds the Mary McDermott Cook Chair in Pediatrics Genetics at UT Southwestern.

Working under the premise that the two findings were pieces of the same puzzle, a crucial part of the picture seemed to be missing, he said.

It was known for decades that the PPP was a major source of NADPH, which provides a source of reducing equivalents (that is, electrons) to scavenge ROS; however, the PPP produces NADPH in a part of the cell called the cytosol, whereas the reactive oxygen species are generated primarily in another subcellular structure called the mitochondria.

“If you think of ROS as fire, then NADPH is like the water used by cancer cells to douse the flames,” Dr. DeBerardinis said. But how could NADPH from the PPP help deal with the stress of ROS produced in a completely different part of the cell? “What we did was to discover how this happens,” Dr. DeBerardinis said.

The current study in Nature demonstrates that cancer cells use a “piggybacking” system to carry reducing equivalents from the PPP into the mitochondria. This movement involves an unusual reaction in the cytosol that transfers reducing equivalents from NADPH to a molecule called citrate, similar to a reversed reaction of the Krebs cycle, he said. The citrate then enters the mitochondria and stimulates another pathway that results in the release of reducing equivalents to produce NADPH right at the location of ROS creation, allowing the cancer cells to survive and grow without the benefit of matrix attachment.

“We knew that both the PPP and Krebs cycle provide metabolic benefits to cancer cells.  But we had no idea that they were linked in this unusual fashion,” he said. “Strikingly, normal cells were unable to transport NADPH by this mechanism, and died as a result of the high ROS levels.”

Dr. DeBerardinis stressed that the findings were based on cultured cell models, and more research will be necessary to test the role of the pathway in living organisms. “We are particularly excited to test whether this pathway is required for metastasis, because cancer cells need to survive in a matrix-detached state in the circulation in order to metastasize,” he said.

CRI scientists find novel metabolic twist that drives cancer survival
http://www.utsouthwestern.edu/newsroom/news-releases/year-2016/april/cancer-metabolism.html

Programmed Cell Death and Cancer Therapy | Leaders in …

https://pharmaceuticalintelligence.com/2016/04/09/programmed-cell-death-and-cancer-therapy/5 days ago … Cancer Cell Survival Driven by Novel Metabolic Pathway … This new study describes an alternate version of two well–known metabolic pathways, the pentose phosphate pathway (PPP) and the Krebs cycle,…

Reductive carboxylation supports growth in tumour cells with …

http://www.nature.com/nature/journal/v481/n7381/abs/nature10642.html Jan 19, 2012 … Nature | Letter …. DeBerardinis, R. J. et al. Beyond aerobic …. Andrew R. Mullen,; Pei-Hsuan Chen,; Tzuling Cheng &; Ralph J. DeBerardinis ..   

Haematopoietic stem cells require a highly regulated protein – Nature
http://www.nature.com/nature/journal/v509/n7498/abs/nature13035.html May 1, 2014 … Nature | Article. Print; Share/ ….. synthesis. Nature Methods 6, 275–277 (2009) …. Robert A. J. Signer,; Jeffrey A. Magee &; Sean J. Morrison …       

Oxidative stress inhibits distant metastasis by human … – Nature

http://www.nature.com/nature/journal/v527/n7577/abs/nature15726.html Nov 12, 2015 … Nature | Article ….. Multistep nature of metastatic inefficiency: dormancy of solitary cells after successful extravasation and ….. Sean J. Morrison …     

Deep imaging of bone marrow shows non-dividing stem … – Nature
http://www.nature.com/nature/journal/v526/n7571/abs/nature15250.html   Oct 1, 2015 … Nature | Letter. Print; Share/ …… Morrison, S. J. & Scadden, D. T. The bone marrow niche for ….. Kiranmai S. Kocherlakota &; Sean J. Morrison …

Genomic Expression | Leaders in Pharmaceutical Business …

https://pharmaceuticalintelligence.com/category/cancer-biology-innovations-in-cancer-therapy/genomic-expression/Glutamine and cancer: cell biology, physiology, and clinical opportunities …. Metabolism of glutamine-derived α-ketoglutarate in the TCA cycle serves … known as hexosamines, that are used to glycosylate growth factor receptors and …… of two well–known metabolic pathways, the pentose phosphate pathway (PPP )

The Mitochondrial Warburg Effect: A Cancer Enigma – IBC Journal
http://www.ibc7.org/article/file_down.php?pid=48&mode=article This feature of cancer cells is known as the Warburg effect, named … new paradigm of collaboration and a well-designed systemic approach will supply … Krebs cycle. … The pentose phosphate pathway uses glucose to produce ribose, which is used … glucose is taken up into cells, it is used in two main metabolic pathways

New paper offers intriguing insights into tumor metabolism     William G. Gilroy    August 19, 2009

Posted In: Research

A paper appearing in this week’s edition of the journal Nature by a team of researchers that includes University of Notre Dame biologist Zachary T. Schafer has important new implications for understanding the metabolism of tumors.

Schafer, an assistant professor of biological sciences and Coleman Junior Chair of Cancer Biology, points out that in the early stages of tumor formation some cells become detached from their normal cellular matrix. These “homeless” cells tend to develop certain defects that stop them from becoming cancerous. In a process known as apoptosis, these precancerous cells essentially kill themselves, allowing them to be destroyed by immune system cells.

The prevailing wisdom among researchers has been that apoptosis was the only way that cells could die.

In studies conducted prior to the research described in the Nature paper, it was found that even when apoptosis was inhibited in detached, precancerous cells, they still eventually died. Intrigued by these results, a team of researchers led by Joan S. Brugge, Louise Foote Pfieffer Professor of Cell Biology at Harvard Medical School, and Schafer decided to take a closer look.

They report in this week’s Nature paper that they found that even when apoptosis was inhibited in detached cells endowed with a cancer-causing gene, they still were incapable of absorbing glucose, their primary energy source. Additionally, the cells displayed signs of oxidative stress, which is a harmful accumulation of oxygen-derived molecules called reactive oxygen species (ROS). The research also revealed decreased ATP production, a key factor in energy transport in the cells.

Schafer notes that this combination of loss of glucose transport, decreased ATP production and heightened oxidative stress reveal a manner of cell death that hadn’t been previously demonstrated to play a role in this context.

In the next phase of the study, Schafer engineered the cells to express a high level of HER2, a gene known to be hyperactive in many breast cancer tumors. He also treated the cells with antioxidants to relieve oxidative stress.

Both approaches helped the cells survive. The HER2-treated cells regained glucose transport, avoided oxidative stress and recovered ATP levels.
Most surprisingly, the antioxidants restored metabolic activity in the cells by allowing fatty acids to be effectively used instead of glucose as an energy source, providing them with a chance to survive.

“Our results raise the possibility that antioxidant activity might allow early stage tumor cells to survive where they would otherwise die from these metabolic defects,” Schafer said.

He also cautions that while the antioxidant findings were surprising, their research was done solely in cell cultures and more research needs to be done before there are clear implications for individuals and their diets.

The paper does, however, offer important new clues about the metabolism of tumor cells and important information that may lead to drugs that can developed to target them.

Antioxidant and Oncogene Rescue of Metabolic Defects Caused by …
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2931797/Aug 19, 2009 …
Nature. Author manuscript; available in PMC 2010 Sep 2. Published in … Y. Irie,¹ Sizhen Gao,¹ Pere Puigserver,^1,2 and Joan S. Brugge^1,*.

Cell Death and Differentiation – Induction of apoptosis in … – Nature

http://www.nature.com/cdd/journal/v10/n8/full/4401251a.html
Proteasome inhibitors have been shown to be effective in cancer treatment, an ability … a specific inhibitor of 26S proteasome, also reduced cell viability ( 80% with 10 mu …
be a consequence of the increased generation of ROS caused by MG132. …. vectors endowed with the wild type forms of RB or p53 genes (Figure 1f).

The Metastasis-Promoting Roles of Tumor-Associated Immune Cells

Heath A. Smith and Yibin Kang
J Mol Med (Berl). 2013 Apr; 91(4): 411–429.     doi:  10.1007/s00109-013-1021-5

Tumor metastasis is driven not only by the accumulation of intrinsic alterations in malignant cells, but also by the interactions of cancer cells with various stromal cell components of the tumor microenvironment. In particular, inflammation and infiltration of the tumor tissue by host immune cells, such as tumor-associated macrophages, myeloid-derived suppressor cells, and regulatory T cells have been shown to support tumor growth in addition to invasion and metastasis. Each step of tumor development, from initiation through metastatic spread, is promoted by communication between tumor and immune cells via the secretion of cytokines, growth factors and proteases that remodel the tumor microenvironment. Invasion and metastasis requires neovascularization, breakdown of the basement membrane, and remodeling of the extracellular matrix for tumor cell invasion and extravasation into the blood and lymphatic vessels. The subsequent dissemination of tumor cells to distant organ sites necessitates a treacherous journey through the vasculature, which is fostered by close association with platelets and macrophages. Additionally, the establishment of the pre-metastatic niche and specific metastasis organ tropism is fostered by neutrophils and bone marrow-derived hematopoietic immune progenitor cells and other inflammatory cytokines derived from tumor and immune cells, which alter the local environment of the tissue to promote adhesion of circulating tumor cells. This review focuses on the interactions between tumor cells and immune cells recruited to the tumor microenvironment, and examines the factors allowing these cells to promote each stage of metastasis.

Once established, tumors are quite adept at preventing anti-tumor immune responses, and several defense mechanisms to circumvent immune detection have been described including antigen loss, down-regulation of major histocompatibility molecules (MHC), deregulation or loss of components of the endogenous antigen presentation pathway, and tumor-induced immune suppression mediated through cytokine secretion or direct interactions between tumor ligands and immune cell receptors [2]. These mechanisms contribute to the process of immunoediting in which tumor cell subpopulations susceptible to immune recognition are lysed and eliminated, while resistant tumor cells proliferate and increase their frequency in the developing neoplasia [3]. However, tumors not only effectively escape immune recognition, they also actively subvert the normal anti-tumor activity of immune cells to promote further tumor growth and metastasis.

During early stages of cancer development, infiltrating immune cell populations are primarily tumor suppressive, but depending on the presence of accessory stromal cells, the local cytokine milieu, and tumor-specific interactions, these immune cells can undergo phenotypic changes to enhance tumor cell dissemination and metastasis. For instance, CD4⁺ T cells, macrophages, and neutrophils have all been shown to possess opposing properties depending on the inflammatory state of the tumor environment, the tissue context, and other cellular stimuli intrinsic to the altered tumor cells [4, 5]. These features are dependent upon the inherent plasticity of immune cells in response to stimulatory or suppressive cytokines [6]. Notably, the switch from a Th1 tumor-suppressive phenotype such as CD4⁺ “helper” T cells, which aid cytotoxic CD8⁺ T cells in tumor rejection, to a Th2 tumor-promoting “regulatory” phenotype, which blocks CD8⁺ T-cell activity, is a characteristic outcome in the inflammatory, immune-suppressive tumor microenvironment [5, 7]. Likewise, M1 macrophages and N1 neutrophils are known to have pronounced anti-tumor activity; however, these immune cells are often subverted to a tumor-promoting M2 and N2 phenotype, respectively, in response to immune-suppressive cytokines secreted by tumor tissue [8].

The crosstalk that occurs between tumor and immune cells within the tumor microenvironment, the circulation, or at distant metastatic sites has been clearly shown to foster metastatic dissemination. Immune cells as well as the suppressive factors that they secrete represent potential targets for therapeutic intervention. Regardless of their source, cytokines, chemokines, proteases, and growth factors are some of the main factors contributing to immunosuppression and immune-mediated tumor progression. These molecules can be produced by immune, stromal, or malignant cells and can act in paracrine and autocrine fashion to promote each stage of tumor cell invasion and metastasis by enhancing inflammation, angiogenesis, tumor proliferation, and recruitment of additional immunosuppressive and tumor-promoting immune cells. These secreted factors provide the malignant cells with an abundant source of growth and survival signals that perpetuate a supportive microenvironment for tumor metastasis and represent some of the most attractive targets for directed anti-tumor therapy. Immune pathways provide numerous soluble targets for cancer treatment, and indeed, many drugs to target immune-suppressive molecules are moving forward in clinical trials. For instance, the anti-RANKL (Denosumab) antibody has been shown to effectively inhibit bone metastasis in prostate cancer patients [201], while a variety of neutralizing antibodies to IL-1β and IL-1 receptor have been shown to have efficacy in treating metastasis in pre-clinical animal models [202]. Several agents that target IL-1 or other immune-suppressive cytokines are already approved for the treatment of some inflammatory diseases and are prime candidates for human trails [202]. Additionally, other proteins involved in tumor progression that are induced directly or indirectly by immune cell populations, such as EMT-associated transcription factors, adhesion molecules, and tumor receptors and ligands which mediate immune suppression, could also be targeted with small molecules or blocking antibodies. Antibodies against two surface molecules expressed by suppressive lymphoid cells, anti-CTLA-4 (ipiliumimab) [203, 204] and anti-PD-1 have been recently gaining increasing support from clinical trials for their effective treatment for many forms of cancer including advanced melanoma and prostate cancer [205, 206]. Specifically, anti-CTLA-4 has been shown to be particularly efficacious in metastatic melanoma, while anti-PD-1 has only just begun a comprehensive evaluation in clinical trials [204, 207]. Likewise, non-steroidal anti-inflammatory drugs (NSAIDS) to prevent or treat chronic inflammation and lymphangiogenesis [208–210], and anti-coagulants to prevent platelet aggregation on circulating tumor cells [211] are just two examples of a multitude of therapeutic agents that could be utilized to prevent immune-mediated tumor progression at unique stages of metastasis. Of course, new methods or biomarkers for the detection of patients at risk of tumor progression or metastasis are also desperately needed to tailor personalized therapy for patients to obtain the best possible clinical outcome.

1. Cancer and Therapeutics | Leaders in Pharmaceutical Business …

   https://pharmaceuticalintelligence.com/category/cancer-and-therapeutics/Mar 26, 2016 … This turns your immune systems ability to attack and kill cancer cells back on” …. the rare skin cancer cells that were able to detach from theprimary tumor and successfully metastasize to other parts of the body had the ability to keep ROS levels from getting dangerously high,” Dr. DeBerardinis remarked.

2. Histone Deacetylase Inhibitors (HDAC) | Leaders in Pharmaceutical …

   https://pharmaceuticalintelligence.com/tag/histone-deacetylase-inhibitors-hdac/The HDAC-inhibiting agent romidepsin significantly increased T-cell tumor … skin cancer cells that were able to detach from the primary tumor and successfully … of the body had the ability to keep ROS levels from getting dangerously high,” Dr. …. Sensitivity for EGFR or KRAS was higher in patients with multiplemetastatic …

3. Genomic Expression | Leaders in Pharmaceutical Business …

   https://pharmaceuticalintelligence.com/category/cancer-biology-innovations-in-cancer-therapy/genomic-expression/In a study involving 320 patients, the researchers were able to infer cell death in …. Glutamine and cancer: cell biology, physiology, and clinical opportunities … On the other hand, GLS2 expression is enhanced in some neuroblastomas, …… of the body had the ability to keep ROS levels from getting dangerously high,” Dr.

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Reactive Oxygen Species and Melanoma

Reactive Oxygen Species and Melanoma

Curator: Larry H Bernstein, MD, FCAP

Antioxidant Use May Promote Melanoma Metastasis

http://www.genengnews.com/gen-news-highlights/antioxidant-use-may-promote-melanoma-metastasis/81251856/

• Click Image To Enlarge +

  Results from a new study suggest that cancer patients should not supplement their diet with large doses of antioxidants. [grThirteen/iStock]

  Decades ago Nobel laureate and American chemist Linus Pauling espoused the benefits of taking megadoses of vitamin C to prevent and treat various diseases. This controversial practice has led many to promote the taking of antioxidant supplements to prevent everything from the common cold to cancer and has become an almost engrained practice for individual healthcare—unfortunately, with little scientific evidence to support the ritual.

  Now, researchers at the Children’s Research Institute at UT Southwestern (CRI) has uncovered evidence that suggests cancer cells benefit more from antioxidants than normal cells, raising concerns about the use of dietary antioxidant supplements by patients with cancer.

  The investigators utilized a specialized mice model that had been transplanted with melanoma cells from patients, which previous work showed recapitulated metastasis of human melanoma cells and was predictive of their metastasis in patients. The CRI team found that when antioxidants were administered to the mice, the cancer spread more quickly than in mice that did not get antioxidants.

  “We discovered that metastasizing melanoma cells experience very high levels of oxidative stress, which leads to the death of most metastasizing cells,” explained senior author Sean Morrison, Ph.D., CRI director and chair in pediatric genetics at UT Southwestern Medical Center. “Administration of antioxidants to the mice allowed more of the metastasizing melanoma cells to survive, increasing metastatic disease burden.”

  The findings from this study were published online today in Nature through an article entitled “Oxidative stress inhibits distant metastasis by human melanoma cells.”

  It has long been known that the spread of cancer cells from one part of the body to another is an inefficient process in which the vast majority of cancer cells that enter the blood fail to survive, due to the highly oxidative environment and exposure to immune cells.

  “The idea that antioxidants are good for you has been so strong that there have been clinical trials done in which cancer patients were administered antioxidants,” noted Dr. Morrison. “Some of those trials had to be stopped because the patients getting the antioxidants were dying faster. Our data suggest the reason for this: cancer cells benefit more from antioxidants than normal cells do.”

  The CRI researchers were intrigued by their findings and acknowledged that although the study’s results have not yet been tested in people, they surmise that cancer should be treated with pro-oxidants and that cancer patients should not supplement their diet with large doses of antioxidants.

  “This finding also opens up the possibility that when treating cancer, we should test whether increasing oxidative stress through the use of pro-oxidants would prevent metastasis,” Dr. Morrison stated. “One potential approach is to target the folate pathway that melanoma cells use to survive oxidative stress, which would increase the level of oxidative stress in the cancer cells.”

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Update on mitochondrial function, respiration, and associated disorders

Larry H. Benstein, MD, FCAP, Gurator and writer

http://pharmaceuticalintelligence.com/7/8/2014/Update on mitochondrial function, respiration, and associated disorders

This is a condensed account of very recent published work on respiration and disturbed mitochondrail function.  We know that their is an equilibrium between respiration and autophagy in eukaryotic cells.  The Krebs Cycle produces 32 ATPs in oxidative phosphorylation, which is far more efficient than glycolysis.  There is also a different contribution of mitochondrial metabolism, in the balance, between tissues that are synthetic and those that are catabolic.  This is a subject long understood, essential for cellular energetics, and not adequately explored.

 

Gain-of-Function Mutant p53 Promotes Cell Growth and Cancer Cell Metabolism via Inhibition of AMPK Activation.

Zhou G¹, Wang J², Zhao M², Xie TX², Tanaka N², et al.
Mol Cell. 2014 Jun 19;54(6):960-974.   doi: 10.1016/j.molcel.2014.04.024. 

Many mutant p53 proteins (mutp53s) exert oncogenic gain-of-function (GOF) properties, but the mechanisms mediating these functions remain poorly defined.

We show here that GOF mutp53s inhibit AMP-activated protein kinase (AMPK) signaling in head and neck cancer cells.

Conversely, downregulation of GOF mutp53s enhances AMPK activation under energy stress, decreasing the activity of the anabolic factors acetyl-CoA carboxylase and ribosomal protein S6 and inhibiting aerobic glycolytic potential and invasive cell growth.

Under conditions of energy stress, GOF mutp53s, but not wild-type p53, preferentially bind to the AMPKα subunit and inhibit AMPK activation.

Given the importance of AMPK as an energy sensor and tumor suppressor that inhibits anabolic metabolism, our findings reveal that direct inhibition of AMPK activation is an important mechanism through which mutp53s can gain oncogenic function. PMID:24857548

Investigating and Targeting Chronic Lymphocytic Leukemia Metabolism with the HIV Protease Inhibitor Ritonavir and Metformin.

Adekola KU, Aydemir SD, Ma S, Zhou Z, Rosen ST, Shanmugam M.
Leuk Lymphoma. 2014 May 14:1-23.

Chronic Lymphocytic Leukemia (CLL) remains fatal due to the development of resistance to existing therapies. Targeting abnormal glucose metabolism sensitizes various cancer cells to chemotherapy and/or elicits toxicity.

Examination of glucose dependency in CLL demonstrated variable sensitivity to glucose deprivation. Further evaluation of metabolic dependencies of CLL cells resistant to glucose deprivation revealed increased engagement of fatty acid oxidation upon glucose withdrawal.

Investigation of glucose transporter expression in CLL reveals up-regulation of glucose transporter GLUT4. Treatment of CLL cells with HIV protease inhibitor ritonavir, that inhibits GLUT4, elicits toxicity similar to that elicited upon glucose-deprivation.

CLL cells resistant to ritonavir are sensitized by co-treatment with metformin, potentially targeting compensatory mitochondrial complex 1 activity. Ritonavir and metformin have been administered in humans for treatment of diabetes in HIV patients, demonstrating the tolerance of this combination in humans. Our studies strongly substantiate further investigation of FDA approved ritonavir and metformin for CLL.

KEYWORDS:  Basic Biology; Chemotherapeutic approaches; Lymphoid Leukemia; Signal transduction             PMID: 24828872

Utilizing hydrogen sulfide as a novel anti-cancer agent by targeting cancer glycolysis and pH imbalance.

Lee ZW¹, Teo XY, Tay EY, Tan CH, Hagen T, Moore PK, Deng LW.
Br J Pharmacol. 2014 May 15.    doi: 10.1111/bph.12773

Many disparate studies have reported the ambiguous role of hydrogen sulfide (H₂ S) in cell survival. The present study investigated the effect of H₂ S on viability of cancer and non-cancer cells.

Cancer and non-cancer cells were exposed to H₂ S (using sodium hydrosulfide, NaHS and GYY4137) and cell viability was examined by crystal violet assay. We then examined cancer cellular glycolysis process by in vitro enzymatic assays and pH regulator activity. Lastly, intracellular pH (pHi) was determined by ratiometric pHi measurement using BCECF staining.

Continuous, but not single, exposure to H₂ S decreased cell survival more effectively in cancer cells, as compared to non-cancer cells. Slow H₂ S-releasing donor, GYY4137, significantly increased glycolysis leading to overproduction of lactate. H₂ S also decreased anion exchanger and sodium/proton exchanger activity. The combination of increased metabolic acid production and defective pH regulation resulted in an uncontrolled intracellular acidification leading to cancer cell death. In contrast, no significant intracellular acidification or cell death was observed in non-cancer cells.

Low and continuous exposure to H₂ S targets metabolic processes and pH homeostasis in cancer cells, potentially serving as a novel and selective anti-cancer strategy.

KEYWORDS:  cancer cell death; cancer glucose metabolism; hydrogen sulfide; pH homeostasis          PMID: 24827113

Agonism of the 5-Hydroxytryptamine 1F Receptor Promotes Mitochondrial Biogenesis and Recovery from Acute Kidney Injury

Garrett SM, Whitaker RM, Beeson CC, and Schnellmann RG

Center for Cell Death, Injury, and Regeneration, Department of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, Charleston, South Carolina (S.M.G., R.M.W., C.C.B., R.G.S.); and Ralph H. Johnson Veterans Affairs Medical Center, Charleston, South Carolina (R.G.S.)
Address correspondence to: Dr. Rick G. Schnellmann, Department of Drug Discovery and Biomedical Sciences, MUSC, Charleston, SC 29425.
E-mail: schnell@musc.edu

Many acute and chronic conditions, such as acute kidney injury, chronic kidney disease, heart failure, and liver disease, involve mitochondrial dysfunction. Although we have provided evidence that drug-induced stimulation of mitochondrial biogenesis (MB) accelerates mitochondrial and cellular repair, leading to recovery of organ function, only a limited number of chemicals have been identified that induce MB.

The goal of this study was to assess the role of the 5-hydroxytryptamine 1F (5-HT_1F) receptor in MB. Immunoblot and quantitative polymerase chain reaction analyses revealed 5-HT_1F receptor expression in renal proximal tubule cells (RPTC). A MB screening assay demonstrated that two selective 5-HT_1F receptor agonists,

1. LY334370 (4-fluoro-N-[3-(1-methyl-4-piperidinyl)-1H-indol-5-yl]benzamide) and
2. LY344864 (N-[(3R)-3-(dimethylamino)-2,3,4,9-tetrahydro-1H-carbazol-6-yl]-4-fluorobenzamide; 1–100 nM)

increased carbonylcyanide-p-trifluoromethoxyphenylhydrazone–uncoupled oxygen consumption in RPTC, and

• validation studies confirmed both agonists increased mitochondrial proteins  in vitro.
  [e.g., ATP synthase β, cytochrome c oxidase 1 (Cox1), and NADH dehydrogenase (ubiquinone) 1β subcomplex subunit 8 (NDUFB8)]

Small interfering RNA knockdown of the 5-HT_1F receptor

• blocked agonist-induced MB.

Furthermore, LY344864 increased

• peroxisome proliferator–activated receptor (PPAR) coactivator 1-α, Cox1, and
• NDUFB8 transcript levels and
• mitochondrial DNA (mtDNA) copy number

in murine renal cortex, heart, and liver.

Finally, LY344864 accelerated recovery of renal function, as indicated by

• decreased blood urea nitrogen and kidney injury molecule 1 and
• increased mtDNA copy number

following ischemia/reperfusion-induced acute kidney injury (AKI).

In summary, these studies reveal that

• the 5-HT_1F receptor is linked to MB, 5-HT_1F receptor agonism promotes MB in vitro and in vivo, and

5-HT_1F receptor agonism promotes recovery from AKI injury.

Induction of MB through 5-HT_1F receptor agonism represents a new target and approach to treat mitochondrial organ dysfunction.

Footnotes

• Portions of this work have been presented previously: Garrett SM, Wills LP, and Schnellmann RG (2012) Serotonin (5-HT) 1F receptor agonism as a potential treatment for acceleration of recovery from acute kidney injury.American Society of Nephrology Annual Meeting; 2012 Nov 1–4; San Diego, CA.
• dx.doi.org/10.1124/jpet.114.214700.

Ca2+ regulation of mitochondrial function in neurons.

Rueda CB¹, Llorente-Folch I¹, Amigo I¹, Contreras L¹, González-Sánchez P¹, Martínez-Valero P¹, Juaristi I¹, Pardo B¹, Del Arco A², Satrústegui J³

Biochim Biophys Acta. 2014 May 10. pii: S0005-2728(14)00126-1.
doi: 10.1016/j.bbabio.2014.04.010.

Calcium is thought to regulate respiration but it is unclear whether this is dependent on the increase in ATP demand caused by any Ca²⁺ signal or to Ca²⁺ itself.

[Na⁺]_i, [Ca²⁺]_i and [ATP]_i dynamics in intact neurons exposed to different workloads in the absence and presence of Ca²⁺ clearly showed that

• Ca²⁺-stimulation of coupled respiration is required to maintain [ATP]_i levels.

Ca²⁺ may regulate respiration by

1. activating metabolite transport in mitochondria from outer face of the inner mitochondrial membrane, or
2. after Ca²⁺ entry in mitochondria through the calcium uniporter (MCU).

Two Ca²⁺-regulated mitochondrial metabolite transporters are expressed in neurons,

1. the aspartate-glutamate exchanger ARALAR/AGC1/Slc25a12, a component of the malate-aspartate shuttle, with a Kd for Ca²⁺ activation of 300nM, and
2. the ATP-Mg/Pi exchanger SCaMC-3/Slc25a23, with S_0.5 for Ca²⁺ of 300nM and 3.4μM, respectively.

The lack of SCaMC-3 results in a smaller Ca²⁺-dependent stimulation of respiration only at high workloads, as caused by veratridine, whereas

• the lack of ARALAR reduced by 46% basal OCR in intact neurons using glucose as energy source and the Ca²⁺-dependent responses to all workloads (veratridine, K⁺-depolarization, carbachol).

The lack of ARALAR caused a reduction of about 65-70% in the response to the high workload imposed by veratridine, and

• completely suppressed the OCR responses to moderate (K⁺-depolarization) and small (carbachol) workloads,
• effects reverted by pyruvate supply.

For K⁺-depolarization, this occurs in spite of the presence of large [Ca²⁺]_mit signals and increased reduction of mitochondrial NAD(P)H.

These results show that ARALAR-MAS is a major contributor of Ca²⁺-stimulated respiration in neurons

• by providing increased pyruvate supply to mitochondria.

In its absence and under moderate workloads, matrix Ca²⁺ is unable to stimulate pyruvate metabolism and entry in mitochondria suggesting a limited role of MCU in these conditions.

This article was invited for a Special Issue entitled: 18th European Bioenergetic Conference.    Copyright © 2014. Published by Elsevier B.V.

KEYWORDS:  ATP-Mg/Pi transporter; Aspartate–glutamate transporter; Calcium; Calcium-regulated transport; Mitochondrion; Neuronal respiration PMID: 24820519

 

Sestrin2 inhibits uncoupling protein 1 expression through suppressing reactive oxygen species.

Ro SH¹, Nam M², Jang I¹, Park HW¹, Park H¹, Semple IA¹, Kim M¹, et al.
Proc Natl Acad Sci U S A. 2014 May 27;111(21):7849-54.
doi: 10.1073/pnas.1401787111.

Uncoupling protein 1 (Ucp1), which is localized in the mitochondrial inner membrane of mammalian brown adipose tissue (BAT), generates heat by uncoupling oxidative phosphorylation. Upon cold exposure or nutritional abundance, sympathetic neurons stimulate BAT to express Ucp1 to induce energy dissipation and thermogenesis. Accordingly, increased Ucp1 expression reduces obesity in mice and is correlated with leanness in humans.

Despite this significance, there is currently a limited understanding of how Ucp1 expression is physiologically regulated at the molecular level. Here, we describe the involvement of Sestrin2 and reactive oxygen species (ROS) in regulation of Ucp1 expression. Transgenic overexpression of Sestrin2 in adipose tissues inhibited both basal and cold-induced Ucp1 expression in interscapular BAT, culminating in decreased thermogenesis and increased fat accumulation.

Endogenous Sestrin2 is also important for suppressing Ucp1 expression because BAT from Sestrin2(-/-) mice exhibited a highly elevated level of Ucp1 expression. The redox-inactive mutant of Sestrin2 was incapable of regulating Ucp1 expression, suggesting that Sestrin2 inhibits Ucp1 expression primarily through reducing ROS accumulation.

Consistently, ROS-suppressing antioxidant chemicals, such as butylated hydroxyanisole and N-acetylcysteine, inhibited cold- or cAMP-induced Ucp1 expression as well. p38 MAPK, a signaling mediator required for cAMP-induced Ucp1 expression, was inhibited by either Sestrin2 overexpression or antioxidant treatments.

Taken together, these results suggest that Sestrin2 and antioxidants inhibit Ucp1 expression through suppressing ROS-mediated p38 MAPK activation, implying a critical role of ROS in proper BAT metabolism.

KEYWORDS: aging; homeostasis; mouse; β-adrenergic signaling      PMID: 24825887     PMCID:  PMC4040599

Mitochondrial EF4 links respiratory dysfunction and cytoplasmic translation in Caenorhabditis elegans.

Yang F¹, Gao Y¹, Li Z², Chen L³, Xia Z⁴, Xu T⁵, Qin Y⁶
Biochim Biophys Acta. 2014 May 15. pii: S0005-2728(14)00499-X.
doi: 10.1016/j.bbabio.2014.05.353.

How animals coordinate cellular bioenergetics in response to stress conditions is an essential question related to aging, obesity and cancer. Elongation factor 4 (EF4/LEPA) is a highly conserved protein that promotes protein synthesis under stress conditions, whereas its function in metazoans remains unknown.

Here, we show that, in Caenorhabditis elegans, the mitochondria-localized CeEF4 (referred to as mtEF4) affects mitochondrial functions, especially at low temperature (15°C).

At worms’ optimum growing temperature (20°C), mtef4 deletion leads to self-brood size reduction, growth delay and mitochondrial dysfunction.

Transcriptomic analyses show that mtef4 deletion induces retrograde pathways, including mitochondrial biogenesis and cytoplasmic translation reorganization.

At low temperature (15°C), mtef4 deletion reduces mitochondrial translation and disrupts the assembly of respiratory chain supercomplexes containing complex IV.

These observations are indicative of the important roles of mtEF4 in mitochondrial functions and adaptation to stressful conditions.

Copyright © 2014. Published by Elsevier B.V.

KEYWORDS: C. elegans; EF4(LepA/GUF1); Mitochondrial dysfunction; Retrograde pathways; Translation    PMID:  24837196

The metabolite α-ketoglutarate extends lifespan by inhibiting ATP synthase and TOR.

Chin RM¹, Fu X², Pai MY³, Vergnes L⁴, Hwang H⁵, Deng G⁶, Diep S², et al.
Nature  2014 Jun 19;509(7505):397-401. doi: 10.1038/nature13264. 

Metabolism and ageing are intimately linked. Compared with ad libitum feeding, dietary restriction consistently extends lifespan and delays age-related diseases in evolutionarily diverse organisms. Similar conditions of nutrient limitation and genetic or pharmacological perturbations of nutrient or energy metabolism also have longevity benefits.

Recently, several metabolites have been identified that modulate ageing; however, the molecular mechanisms underlying this are largely undefined. Here we show that α-ketoglutarate (α-KG), a tricarboxylic acid cycle intermediate, extends the lifespan of adult Caenorhabditis elegans.

ATP synthase subunit β is identified as a novel binding protein of α-KG using a small-molecule target identification strategy termed drug affinity responsive target stability (DARTS). The ATP synthase, also known as complex V of the mitochondrial electron transport chain, is the main cellular energy-generating machinery and is highly conserved throughout evolution.

Although complete loss of mitochondrial function is detrimental, partial suppression of the electron transport chain has been shown to extend C. elegans lifespan.

We show that α-KG inhibits ATP synthase and, similar to ATP synthase knockdown, inhibition by α-KG leads to reduced ATP content, decreased oxygen consumption, and increased autophagy in both C. elegans and mammalian cells.

We provide evidence that the lifespan increase by α-KG requires ATP synthase subunit β and is dependent on target of rapamycin (TOR) downstream.

Endogenous α-KG levels are increased on starvation and α-KG does not extend the lifespan of dietary-restricted animals, indicating that α-KG is a key metabolite that mediates longevity by dietary restriction.

Our analyses uncover new molecular links between a common metabolite, a universal cellular energy generator and dietary restriction in the regulation of organismal lifespan, thus suggesting new strategies for the prevention and treatment of ageing and age-related diseases.

PMID: 24828042

 

 

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Telling NO to Cardiac Risk

DDAH Says NO to ADMA(1); The DDAH/ADMA/NOS Pathway(2)

Author-Writer-Reporter:  Stephen J. Williams, PhD

Endothelium-derived nitric oxide (NO) has been shown to be vasoprotective.  Nitric oxide enhances endothelial cell survival, inhibits excessive proliferation of vascular smooth muscle cells, regulates vascular smooth muscle tone, and prevents platelets from sticking to the endothelial wall.  Together with evidence from preclinical and human studies, it is clear that impairment of the NOS pathway increases risk of cardiovascular disease (3-5).

This post contains two articles on the physiological regulation of nitric oxide (NO) by an endogenous NO synthase inhibitor asymmetrical dimethylarginine (ADMA) and ADMA metabolism by the enzyme DDAH(1,2).  Previous posts on nitric oxide, referenced at the bottom of the page, provides excellent background and further insight for this posting. In summary plasma ADMA levels are elevated in patients with cardiovascular disease and several large studies have shown that plasma ADMA is an independent biomarker for cardiovascular-related morbidity and mortality(6-8).

Figure 1 A. Cardiac risks of ADMA B. Effects of ADMA (Photo credit: Wikipedia)

ADMA Production and Metabolism

Nuclear proteins such as histones can be methylated on arginine residues by protein-arginine methyltransferases, enzymes which use S-adenosylmethionine as methyl groups.  This methylation event is thought to regulate protein function, much in the way of protein acetylation and phosphorylation (9).  And much like phosphorylation, these modifications are reversible through methylesterases.   The proteolysis of these arginine-methyl modifications lead to the liberation of free guanidine-methylated arginine residues such as L-NMMA, asymmetric dimethylarginine (ADMA) and symmetrical methylarginine (SDMA).

The first two, L-NMMA and ADMA, have been shown to inhibit the activity of the endothelial NOS.  This protein turnover is substantial: for instance the authors note that each day 40% of constitutive protein in adult liver is newly synthesized protein. And in several diseases, such as muscular dystrophy, ischemic heart disease, and diabetes, it has been known since the 1970’s that protein catabolism rates are very high, with corresponding increased urinary excretion of ADMA(10-13).  Methylarginines are excreted in the urine by cationic transport.  However, the majority of ADMA and L-NMMA are degraded within the cell by dimethylaminohydrolase (DDAH), first cloned and purified in rat(14).

Figure 2.  Endogenous inhibitors of NO synthase.  Chemical structures generated from PubChem.

DDAH

DDAH specifically hydrolyzes ADMA and L-NMMA to yield citruline and demethylamine and usually shows co-localization with NOS. Pharmacologic inhibition of DDAH activity causes accumulation of ADMA and can reverse the NO-mediated bradykinin-induced relaxation of human saphenous vein.

Two isoforms have been found in human:

• DDAH1 (found in brain and kidney and associated with nNOS) and
• DDAH2 (highly expressed in heart, placenta, and kidney and associated with eNOS).

DDAH2 can be upregulated by all-trans retinoic acid (atRA can increase NO production).  Increased reactive oxygen species and possibly homocysteine, a risk factor for cardiovascular disease, can decrease DDAH activity(15,16).

• The importance of DDAH activity can also be seen in transgenic mice which overexpress DDAH, exhibiting increased NO production, increased insulin sensitivity, and reduced vascular resistance  (17).  Likewise,
• Transgenic mice, null for the DDAH1, showed increase in blood pressure, decreased NO production, and significant increase in tissue and plasma ADMA and L-NMMA.

Figure 3.  The DDAH/ADMA/NOS cycle. Figure adapted from Cooke and Ghebremarian (1).

As mentioned in the article by Cooke and Ghebremariam, the authors state: the weight of the evidence indicates that DDAH is a worthy therapeutic target. Agents that increase DDAH expression are known, and 1 of these, a farnesoid X receptor agonist, is in clinical trials

http://www.interceptpharma.com

An alternate approach is to

• develop an allosteric activator of the enzyme.  Although
• development of an allosteric activator is not a typical pharmaceutical approach, recent studies indicate that this may be achievable aim(18).

References:

1.            Cooke, J. P., and Ghebremariam, Y. T. : DDAH says NO to ADMA.(2011) Arteriosclerosis, thrombosis, and vascular biology 31, 1462-1464

2.            Tran, C. T., Leiper, J. M., and Vallance, P. : The DDAH/ADMA/NOS pathway.(2003) Atherosclerosis. Supplements 4, 33-40

3.            Niebauer, J., Maxwell, A. J., Lin, P. S., Wang, D., Tsao, P. S., and Cooke, J. P.: NOS inhibition accelerates atherogenesis: reversal by exercise. (2003) American journal of physiology. Heart and circulatory physiology 285, H535-540

4.            Miyazaki, H., Matsuoka, H., Cooke, J. P., Usui, M., Ueda, S., Okuda, S., and Imaizumi, T. : Endogenous nitric oxide synthase inhibitor: a novel marker of atherosclerosis.(1999) Circulation 99, 1141-1146

5.            Wilson, A. M., Shin, D. S., Weatherby, C., Harada, R. K., Ng, M. K., Nair, N., Kielstein, J., and Cooke, J. P. (2010): Asymmetric dimethylarginine correlates with measures of disease severity, major adverse cardiovascular events and all-cause mortality in patients with peripheral arterial disease. Vasc Med 15, 267-274

6.            Kielstein, J. T., Impraim, B., Simmel, S., Bode-Boger, S. M., Tsikas, D., Frolich, J. C., Hoeper, M. M., Haller, H., and Fliser, D. : Cardiovascular effects of systemic nitric oxide synthase inhibition with asymmetrical dimethylarginine in humans.(2004) Circulation 109, 172-177

7.            Kielstein, J. T., Donnerstag, F., Gasper, S., Menne, J., Kielstein, A., Martens-Lobenhoffer, J., Scalera, F., Cooke, J. P., Fliser, D., and Bode-Boger, S. M. : ADMA increases arterial stiffness and decreases cerebral blood flow in humans.(2006) Stroke; a journal of cerebral circulation 37, 2024-2029

8.            Mittermayer, F., Krzyzanowska, K., Exner, M., Mlekusch, W., Amighi, J., Sabeti, S., Minar, E., Muller, M., Wolzt, M., and Schillinger, M. : Asymmetric dimethylarginine predicts major adverse cardiovascular events in patients with advanced peripheral artery disease.(2006) Arteriosclerosis, thrombosis, and vascular biology 26, 2536-2540

9.            Kakimoto, Y., and Akazawa, S.: Isolation and identification of N-G,N-G- and N-G,N’-G-dimethyl-arginine, N-epsilon-mono-, di-, and trimethyllysine, and glucosylgalactosyl- and galactosyl-delta-hydroxylysine from human urine. (1970) The Journal of biological chemistry 245, 5751-5758

10.          Inoue, R., Miyake, M., Kanazawa, A., Sato, M., and Kakimoto, Y.: Decrease of 3-methylhistidine and increase of NG,NG-dimethylarginine in the urine of patients with muscular dystrophy. (1979) Metabolism: clinical and experimental 28, 801-804

11.          Millward, D. J.: Protein turnover in skeletal muscle. II. The effect of starvation and a protein-free diet on the synthesis and catabolism of skeletal muscle proteins in comparison to liver. (1970) Clinical science 39, 591-603

12.          Goldberg, A. L., and St John, A. C.: Intracellular protein degradation in mammalian and bacterial cells: Part 2. (1976) Annual review of biochemistry 45, 747-803

13.          Dice, J. F., and Walker, C. D.: Protein degradation in metabolic and nutritional disorders. (1979) Ciba Foundation symposium, 331-350

14.          Ogawa, T., Kimoto, M., and Sasaoka, K.: Purification and properties of a new enzyme, NG,NG-dimethylarginine dimethylaminohydrolase, from rat kidney. (1989) The Journal of biological chemistry 264, 10205-10209

15.          Ito, A., Tsao, P. S., Adimoolam, S., Kimoto, M., Ogawa, T., and Cooke, J. P.: Novel mechanism for endothelial dysfunction: dysregulation of dimethylarginine dimethylaminohydrolase. (1999) Circulation 99, 3092-3095

16.          Stuhlinger, M. C., Tsao, P. S., Her, J. H., Kimoto, M., Balint, R. F., and Cooke, J. P. : Homocysteine impairs the nitric oxide synthase pathway: role of asymmetric dimethylarginine.(2001) Circulation 104, 2569-2575

17.          Sydow, K., Mondon, C. E., Schrader, J., Konishi, H., and Cooke, J. P.: Dimethylarginine dimethylaminohydrolase overexpression enhances insulin sensitivity. (2008) Arteriosclerosis, thrombosis, and vascular biology 28, 692-697

18.          Zorn, J. A., and Wells, J. A.: Turning enzymes ON with small molecules. (2010) Nature chemical biology 6, 179-188

Other research papers on Nitric Oxide and Cardiac Risk  were published on this Scientific Web site as follows:

The Nitric Oxide and Renal is presented in FOUR parts:

Part I: The Amazing Structure and Adaptive Functioning of the Kidneys: Nitric Oxide

Part II: Nitric Oxide and iNOS have Key Roles in Kidney Diseases

Part III: The Molecular Biology of Renal Disorders: Nitric Oxide

Part IV: New Insights on Nitric Oxide donors

Cardiac Arrhythmias: A Risk for Extreme Performance Athletes

What is the role of plasma viscosity in hemostasis and vascular disease risk?

Cardiovascular Risk Inflammatory Marker: Risk Assessment for Coronary Heart Disease and Ischemic Stroke – Atherosclerosis.

Endothelial Dysfunction, Diminished Availability of cEPCs, Increasing CVD Risk for Macrovascular Disease – Therapeutic Potential of cEPCs

Biochemistry of the Coagulation Cascade and Platelet Aggregation – Part I

Nitric Oxide Function in Coagulation

Nitric Oxide, Platelets, Endothelium and Hemostasis

NO Nutritional remedies for hypertension and atherosclerosis. It’s 12 am: do you know where your electrons are?

Endothelin Receptors in Cardiovascular Diseases: The Role of eNOS Stimulation

Nitric Oxide and Sepsis, Hemodynamic Collapse, and the Search for Therapeutic Options

Nitric Oxide and Immune responses: part 1

Interaction of Nitric Oxide and Prostacyclin in Vascular Endothelium

Crucial role of Nitric Oxide in Cancer

Nitric oxide: role in Cardiovascular health and disease

Nitric Oxide Covalent Modifications: A Putative Therapeutic Target?

Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function

Nitric Oxide Signalling Pathways

Statins’ nonlipid effects on vascular endothelium through eNOS activation

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Mitochondrial fission and fusion: potential therapeutic targets?

Author and Curator: Ritu Saxena, Ph.D.

Introduction: Mitochondrial fission & fusion

Mitochondria, double membranous and semi-autonomous organelles, are known to convert energy into forms that are usable to the cell. Apart from being sites of cellular respiration, multiple roles of mitochondria have been emphasized in processes such as cell division, growth and cell death. Mitochondria are semi-autonomous in that they are only partially dependent on the cell to replicate and grow. They have their own DNA, ribosomes, and can make their own proteins. Mitochondria have been discussed in several posts published in the Pharmaceutical Intelligence blog.

Mitochondria do not exist as lone organelles, but are part of a dynamic network that continuously undergoes fusion and fission in response to various metabolic and environmental stimuli. Nucleoids, the assemblies of mitochondrial DNA (mtDNA) with its associated proteins, are distributed during fission in such a way that each mitochondrion contains at least one nucleoid. Mitochondrial fusion and fission within a cell is speculated to be involved in several functions including mtDNA DNA protection, alteration of cellular energetics, and regulation of cell division.

Proteins involved in mitochondrial fission & fusion

Multiple mitochondrial membrane GTPases that regulate mitochondrial networking have recently been identified. They are classified as fission and fusion proteins:

Fusion proteins: Members of dynamin family of protein, mitofusin 1 (Mfn-1) and mitofusin 2 (Mfn-2), are involved in fusion between mitochondria by tethering adjacent mitochondria. These proteins have two transmembrane segments that anchor them in the mitochondrial outer membrane. Mutations in Mitofusin proteins gives rise to fragmented mitochondria, but this can be reversed by mutations in mammalian Drp1. Mitochondrial inner membranes are fused by dynamin family members called Opa1.

Fission proteins: Another member of the dynamin family of proteins, dynamin-related protein 1 (Drp-1) mediates fission of mitochondria. Drp-1 is activated by phosphorylation. Drp-1 proteins are largely cytosolic, but cycle on and off of mitochondria as needed for fission. Fission is a complex process and involves a series of well-defined stages and proteins. Cytosolic Drp-1 is activated by calcineurin or other cytosolic signaling proteins after which it translocates to the mitochondrial tubules where it assembles into foci through its interaction with another protein, hFis1. Once Drp-1 rings assemble on the constricted spots, outer membrane of mitochondria undergoes fission through GTP hydrolysis. Drp-1 is now left bound to one of the newly formed mitochondrial ends after which it slowly disassembles before returning to the cytoplasm.

Control of mitochondrial fission & fusion

• Mitochondrial fission and fusion are controlled by several regulatory mechanisms. Few of which are mentioned as follows:
• Drp-1 activation by Cdk1/Cyclin B mediated phosphorylation during mitosis – triggers fission
• Drp-1 inactivation by cAMP-dependent protein kinase (PKA) in quiescent cells- prevents fission
• Drp-1 activation after reversal of PKA phosphorylation by Calcineurin- triggers fission
• Ubiquination of fission and fusion proteins by E3 ubiquitin ligase- alters fission
• Sumoylation of fission proteins – regulates fission

Imparied mitochondrial fission leads to loss of mtDNA

Mitochondrial fission plays an important role in mitochondrial and cellular homeostasis. It was reported by Parone et al (2008) that preventing mitochondrial fission by down-regulating expression of Drp-1 lead to loss of mtDNA and mitochondrial dysfunction. An increase in cellular reactive oxygen species (ROS) was observed. Other cellular implications included depletion of cellular ATP, inhibition of cell proliferation and autophagy. The observations were made in HeLa cells.

MicroRNA regulation of mitochondrial fission

Although several factors have been attributed to the regulation of mitochondrial fission, the mechanism still remains poorly understood. Recently, regulation of mitochondrial fission via miRNAs has become a topic of interest. Following miRNAs have been found to be involved in mitochondrial fission:

• miR-484:  Wang et al (2012) demonstrated that miR-484 was able to regulate mitochondrial fission by suppressing the translation of a fission protein Fis1, leading to inhibition of Fis1-mediated fission and apoptosis in cardiomyocytes and in the adrenocortical cancer cells. The authors showed that Fis1 is necessary for mitochondrial fission and apoptosis, and is upregulated during anoxia, whereas miR-484 is downregulated. Underlying mechanism involved transactivation of miR-484 by a transcription factor, Foxo3a and miR-484 is able to attenuate Fis1 upregulation and mitochondrial fission, by binding to the amino acid coding sequence of Fis1 and inhibiting its translation.
• miR-499: miR-499 was reported by Wang et al (2011) to be able to directly target both the α- and β-isoforms of the calcineurin catalytic subunit. Suppression of calcineurin-mediated dephosphorylation of  Drp-1 lead to inhibition of the fission machinery ultimately resulting in the inhibition of cardiomyocyte apoptosis. miR-499 levels, by altering mitochondrial fusion were able affect the severity of myocardial infarction and cardiac dysfunction induced by ischemia-reperfusion. Modulation of miR-499 expression could provide a therapeutic approach for myocardial infarction treatment.
• miR-30: It was reported by Li et al (2010) that miR-30 family members were able to inhibit mitochondrial fission and also the resulting apoptosis. While exploring the underlying molecular mechanism, the authors identified that miR-30 family members can suppress p53 expression. When cell received apoptotic stimulation, p53 was found to transcriptionally activate the fission protein, Drp-1. Drp-1 was able to induce mitochondrial fission. miR-30 family members were observed to inhibit mitochondrial fission through attenuation of p53 expression and its downstream target Drp-1.

Mitochondrial fission & fusion as a therapeutic target

Since alteration of mitochondrial fission and fusion have been reported to affect various cellular processes including apoptosis, proliferation, ATP consumption, the proteins involved in the process of fission and fusion might be harnessed as therapeutic target.

Mentioned below is a description of research where dynamics of the mitochondrial organelle has been utilized as a therapeutic target:

Inhibition of mitochondrial fission prevents cell cycle progression in lung cancer

A recent article published by Rehman et al (2012) in the FASEB journal drew much attention after interesting observations were made in the mitochondria of lung adenocarcinoma cells. The mitochondrial network of these cells exhibited both impaired fusion and enhanced fission. It was also found that the fragmented phenotype in multiple lung adenocarcinoma cell lines was associated with both a down-regulation of the fusion protein, Mfn-2 and an upregulation of expression of fission protein, Drp-1. The imbalance of Drp-1/Mfn-2 expression in human lung cancer cell lines was reported to promote a state of mitochondrial fission. Similar increase in Drp-1 and decrease in Mfn-2 was observed in the tissue samples from patients compared to adjacent healthy lung. Authors used complementary approaches of Mfn-2 overexpression, Drp-1 inhibition, or Drp-1 knockdown and were able to observe reduction of cancer cell proliferation and an increase spontaneous apoptosis. Thus, the study identified mitochondrial fission and Drp-1 activation as a novel therapeutic target in lung cancer.

Reference:

Research articles-

http://www.ncbi.nlm.nih.gov/pubmed/20556877

http://www.ncbi.nlm.nih.gov/pubmed?term=18806874

http://www.ncbi.nlm.nih.gov/pubmed/22510686

http://www.ncbi.nlm.nih.gov/pubmed/21186368

http://www.ncbi.nlm.nih.gov/pubmed?term=20062521

http://www.ncbi.nlm.nih.gov/pubmed?term=22321727

News brief:

http://www.uchospitals.edu/news/2012/20120221-mitochondria.html

http://news.uchicago.edu/article/2012/02/23/energy-network-within-cells-may-be-new-target-cancer-therapy

http://www.doctortipster.com/7881-mitochondria-could-represent-a-new-target-for-cancer-therapy-according-to-new-study.html

Related reading:

Reviewer: Larry H Bernstein, MD, FACP

http://pharmaceuticalintelligence.com/2012/10/28/mitochondrial-damage-and-repair-under-oxidative-stress/

Author and Curator: Larry H Bernstein, MD, FACP http://pharmaceuticalintelligence.com/2012/09/26/mitochondria-origin-from-oxygen-free-environment-role-in-aerobic-glycolysis-metabolic-adaptation/

Reporter and Editor: Larry H Bernstein, MD, FACP

http://pharmaceuticalintelligence.com/2012/09/16/nitric-oxide-has-a-ubiquitous-role-in-the-regulation-of-glycolysis-with-a-concomitant-influence-on-mitochondrial-function/

Author and Reporter: Ritu Saxena, PhD

http://pharmaceuticalintelligence.com/2012/09/10/%CE%B2-integrin-emerges-as-an-important-player-in-mitochondrial-dysfunction-associated-gastric-cancer/

Author: Ritu Saxena, PhD

http://pharmaceuticalintelligence.com/2012/09/01/mitochondria-and-cancer-an-overview/

Author and Reporter: Ritu Saxena, PhD

http://pharmaceuticalintelligence.com/2012/08/14/mitochondrial-mutation-analysis-might-be-1-step-away/

Reporter: Venkat S. Karra, PhD

http://pharmaceuticalintelligence.com/2012/08/14/detecting-potential-toxicity-in-mitochondria/

Reporter: Aviva Lev-Ari, PhD, RN http://pharmaceuticalintelligence.com/2012/08/01/mitochondrial-mechanisms-of-disease-in-diabetes-mellitus/

Author and Curator: Ritu Saxena, PhD; Consultants: Aviva Lev-Ari, PhD, RN and Pnina G. Abir-Am, PhD

http://pharmaceuticalintelligence.com/2012/07/09/mitochondria-more-than-just-the-powerhouse-of-the-cell/

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Nitric Oxide and Immune Responses: Part 2

Curator/Reporter Aviral Vatsa PhD, MBBS

Based on: A review by (Wink et al., 2011)

This post is in continuation to Part 1 by the same title.

In part one I covered the basics of role of redox chemistry in immune reactions, the phagosome cauldron, and how bacteria bacteria, virus and parasites trigger the complex pathway of NO production and its downstream effects. While we move further in this post, the previous post can be accessed here.

REDOX REGULATION OF IMMUNE FUNCTION

Regulation of the redox immunomodulators—NO/RNS and ROS

In addition to eradicating pathogens, NO/RNS and ROS and their chemical interactions act as effective immunomodulators that regulate many cellular metabolic pathways and tissue repair and proinflammatory pathways. Figure 3 shows these pathways.

Figure 3. Schematic overview of interactive connections between NO and ROS-mediated metabolic pathways. Credit: (Wink et al., 2011)

Regulation of iNOS enzyme activity is critical to NO production. Factors such as the availability of arginine, BH4, NADPH, and superoxide affect iNOS activity and thus NO production. In the absence of arginine and BH4 iNOS becomes a O2_/H2O2 generator (Vásquez-Vivar et al., 1999). Hence metabolic pathways that control arginine and BH4 play a role in determining the NO/superoxide balance. Arginine levels in cells depend on various factors such as type of uptake mechanisms that determine its spatial presence in various compartments and enzymatic systems. As shown in Fig3 Arginine is the sole substrate for iNOS and arginase. Arginase is another key enzyme in immunemodulation. AG is also regulated by NOS and NOX activities. NOHA, a product of NOS, inhibits AG, and O2–increases AG activity. Importantly, high AG activity is associated with elevated ROS and low NO fluxes. NO antagonises NOX2 assembly that in turn leads to reduction in O2_ production. NO also inhibits COX2 activity thus reducing ROS production. Thus, as NO levels decline, oxidative mechanisms increase. Oxidative and nitrosative stress can also decrease intracellular GSH (reduced form) levels, resulting in a reduced antioxidant capability of the cell.

Immune-associated redox pathways regulate other important metabolic cell functions that have the potential for widespread impact on cells, organs, and organisms. These pathways, such as mediated via methionine and polyamines, are critical for DNA stabilization, cell proliferation, and membrane channel activity, all of which are also involved in immune-mediated repair processes.

NO levels dictate the immune signaling pathway

NO/RNS and ROS actively control innate and adaptive immune signaling by participating in induction, maintenance, and/or termination of proinflammatory and anti-inflammatory signaling. As in pathogen eradication, the temporal and spatial concentration profiles of NO are key factors in determining immune-mediated processes.

Brune and coworkers (Messmer et al., 1994) first demonstrated that p53 expression was associated with the concentrations of NO that led to apoptosis in macrophages. Subsequent studies linked NO concentration profiles with expression of other key signaling proteins such as HIF-1α and Akt-P (Ridnour et al., 2008; Thomas et al., 2008). Various levels of NO concentrations trigger different pathways and expectedly this concentration-dependent profile varies with distance from the NO source.NO is highly diffucible and this characteristic can result in 1000 fold reduction in concentration within one cell length distance travelled from the source of production. Time course studies have also shown alteration in effects of same levels of NO over time e.g. NO-mediated ERK-P levels initially increased rapidly on exposure to NO donors and then decreased with continued NO exposure (Thomas et al., 2004), however HIF-1α levels remained high as long as NO levels were elevated. Thus some of the important factors that play critical role in NO effects are: distance from source, NO concentrations, duration of exposure, bioavailability of NO, and production/absence of other redox molecules.

Figure and legend credits: (Wink et al., 2011)

Fig 4: The effect of steady-state flux of NO on signal transduction mechanisms.

This diagram represents the level of sustained NO that is required to activate specific pathways in tumor cells. Similar effects have been seen on endothelial cells. These data were generated by treating tumor or endothelial cells with the NO donor DETANO (NOC-18) for 24 h and then measuring the appropriate outcome measures (for example, p53 activation). Various concentrations of DETANO that correspond to cellular levels of NO are: 40–60 μM DETANO = 50 nM NO; 80–120 μM DETANO = 100 nM NO; 500 μM DETANO = 400 nM NO; and 1 mM DETANO = 1 μM NO. The diagram represents the effect of diffusion of NO with distance from the point source (an activated murine macrophage producing iNOS) in vitro (Petri dish) generating 1 μM NO or more. Thus, reactants or cells located at a specific distance from the point source (i.e., iNOS, represented by star) would be exposed to a level of NO that governs a specific subset of physiological or pathophysiological reactions. The x-axis represents the different zone of NO-mediated events that is experienced at a specific distance from a source iNOS producing >1 μM. Note: Akt activation is regulated by NO at two different sites and by two different concentration levels of NO.

Species-specific NO production

The relationship of NO and immunoregulation has been established on the basis of studies on tumor cell lines or rodent macrophages, which are readily available sources of NO. However in humans the levels of protein expression for NOS enzymes and the immune induction required for such levels of expression are quite different than in rodents (Weinberg, 1998). This difference is most likely due to the human iNOS promotor rather than the activity of iNOS itself. There is a significant mismatch between the promoters of humans and rodents and that is likely to account for the notable differences in the regulation of gene induction between them. The combined data on rodent versus human NO and O2– production strongly suggest that in general, ROS production is a predominant feature of activated human macrophages, neutrophils, and monocytes, and the equivalent murine immune cells generate a combination of O2– and NO and in some cases, favor NO production. These differences may be crucial to understanding how immune responses are regulated in a species-specific manner. This is particularly useful, as pathogen challenges change constantly.

The next post in this series will cover the following topics:

The impact of NO signaling on an innate immune response—classical activation

NO and proinflammatory genes

NO and regulation of anti-inflammatory pathways

NO impact on adaptive immunity—immunosuppression and tissue-restoration response

NO and revascularization

Acute versus chronic inflammatory disease

Bibliography

1. Wink, D. A. et al. Nitric oxide and redox mechanisms in the immune response. J Leukoc Biol 89, 873–891 (2011).

2. Vásquez-Vivar, J. et al. Tetrahydrobiopterin-dependent inhibition of superoxide generation from neuronal nitric oxide synthase. J. Biol. Chem. 274, 26736–26742 (1999).

3. Messmer, U. K., Ankarcrona, M., Nicotera, P. & Brüne, B. p53 expression in nitric oxide-induced apoptosis. FEBS Lett. 355, 23–26 (1994).

4. Ridnour, L. A. et al. Molecular mechanisms for discrete nitric oxide levels in cancer. Nitric Oxide 19, 73–76 (2008).

5. Thomas, D. D. et al. The chemical biology of nitric oxide: implications in cellular signaling. Free Radic. Biol. Med. 45, 18–31 (2008).

6. Thomas, D. D. et al. Hypoxic inducible factor 1alpha, extracellular signal-regulated kinase, and p53 are regulated by distinct threshold concentrations of nitric oxide. Proc. Natl. Acad. Sci. U.S.A. 101, 8894–8899 (2004).

7. Weinberg, J. B. Nitric oxide production and nitric oxide synthase type 2 expression by human mononuclear phagocytes: a review. Mol. Med. 4, 557–591 (1998).

Further reading on NO:

Nitric Oxide in bone metabolism July 16, 2012

Author: Aviral Vatsa PhD, MBBS

http://pharmaceuticalintelligence.com/2012/07/16/nitric-oxide-in-bone-metabolism/?goback=%2Egde_4346921_member_134751669

Nitric Oxide production in Systemic sclerosis July 25, 2012

Curator: Aviral Vatsa, PhD, MBBS

http://pharmaceuticalintelligence.com/2012/07/25/nitric-oxide-production-in-systemic-sclerosis/?goback=%2Egde_4346921_member_138370383

Nitric Oxide Signalling Pathways August 22, 2012 by

Curator/ Author: Aviral Vatsa, PhD, MBBS

http://pharmaceuticalintelligence.com/2012/08/22/nitric-oxide-signalling-pathways/?goback=%2Egde_4346921_member_151245569

Nitric Oxide: a short historic perspective August 5, 2012

Author/Curator: Aviral Vatsa PhD, MBBS

http://pharmaceuticalintelligence.com/2012/08/05/nitric-oxide-a-short-historic-perspective-7/

Nitric Oxide: Chemistry and function August 10, 2012

Curator/Author: Aviral Vatsa PhD, MBBS

http://pharmaceuticalintelligence.com/2012/08/10/nitric-oxide-chemistry-and-function/?goback=%2Egde_4346921_member_145137865

Nitric Oxide and Platelet Aggregation August 16, 2012 by

Author: Dr. Venkat S. Karra, Ph.D.

http://pharmaceuticalintelligence.com/2012/08/16/no-and-platelet-aggregation/?goback=%2Egde_4346921_member_147475405

The rationale and use of inhaled NO in Pulmonary Artery Hypertension and Right Sided Heart Failure August 20, 2012

Author: Larry Bernstein, MD

http://pharmaceuticalintelligence.com/2012/08/20/the-rationale-and-use-of-inhaled-no-in-pulmonary-artery-hypertension-and-right-sided-heart-failure/

Nitric Oxide: The Nobel Prize in Physiology or Medicine 1998 Robert F. Furchgott, Louis J. Ignarro, Ferid Murad August 16, 2012

Reporter: Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2012/08/16/nitric-oxide-the-nobel-prize-in-physiology-or-medicine-1998-robert-f-furchgott-louis-j-ignarro-ferid-murad/

Coronary Artery Disease – Medical Devices Solutions: From First-In-Man Stent Implantation, via Medical Ethical Dilemmas to Drug Eluting Stents August 13, 2012

Author: Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2012/08/13/coronary-artery-disease-medical-devices-solutions-from-first-in-man-stent-implantation-via-medical-ethical-dilemmas-to-drug-eluting-stents/

Nano-particles as Synthetic Platelets to Stop Internal Bleeding Resulting from Trauma

August 22, 2012

Reported by: Dr. V. S. Karra, Ph.D.

http://pharmaceuticalintelligence.com/2012/08/22/nano-particles-as-synthetic-platelets-to-stop-internal-bleeding-resulting-from-trauma/

Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production July 19, 2012

Curator and Research Study Originator: Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2012/07/19/cardiovascular-disease-cvd-and-the-role-of-agent-alternatives-in-endothelial-nitric-oxide-synthase-enos-activation-and-nitric-oxide-production/

Macrovascular Disease – Therapeutic Potential of cEPCs: Reduction Methods for CV Risk

July 2, 2012

An Investigation of the Potential of circulating Endothelial Progenitor Cells (cEPCs) as a Therapeutic Target for Pharmacological Therapy Design for Cardiovascular Risk Reduction: A New Multimarker Biomarker Discovery

Curator: Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2012/07/02/macrovascular-disease-therapeutic-potential-of-cepcs-reduction-methods-for-cv-risk/

Bone remodelling in a nutshell June 22, 2012

Author: Aviral Vatsa, Ph.D., MBBS

http://pharmaceuticalintelligence.com/2012/06/22/bone-remodelling-in-a-nutshell/

Targeted delivery of therapeutics to bone and connective tissues: current status and challenges- Part, September  

Author: Aviral Vatsa, PhD, September 23, 2012

http://pharmaceuticalintelligence.com/2012/09/23/targeted-delivery-of-therapeutics-to-bone-and-connective-tissues-current-status-and-challenges-part-i/

Calcium dependent NOS induction by sex hormones: Estrogen

Curator: S. Saha, PhD, October 3, 2012

http://pharmaceuticalintelligence.com/2012/10/03/calcium-dependent-nos-induction-by-sex-hormones/

Nitric Oxide and Platelet Aggregation,

Author V. Karra, PhD, August 16, 2012

http://pharmaceuticalintelligence.com/2012/08/16/no-and-platelet-aggregation/

Bystolic’s generic Nebivolol – positive effect on circulating Endothelial Progenitor Cells endogenous augmentation

Curator: Aviva Lev-Ari, PhD, July 16, 2012

http://pharmaceuticalintelligence.com/?s=Nebivolol

Endothelin Receptors in Cardiovascular Diseases: The Role of eNOS Stimulation

Author: Aviva Lev-Ari, PhD, 10/4/2012

http://pharmaceuticalintelligence.com/2012/10/04/endothelin-receptors-in-cardiovascular-diseases-the-role-of-enos-stimulation/

Inhibition of ET-1, ETA and ETA-ETB, Induction of NO production, stimulation of eNOS and Treatment Regime with PPAR-gamma agonists (TZD): cEPCs Endogenous Augmentation for Cardiovascular Risk Reduction – A Bibliography

Curator: Aviva Lev-Ari, 10/4/2012.

http://pharmaceuticalintelligence.com/2012/10/04/inhibition-of-et-1-eta-and-eta-etb-induction-of-no-production-and-stimulation-of-enos-and-treatment-regime-with-ppar-gamma-agonists-tzd-cepcs-endogenous-augmentation-for-cardiovascular-risk-reduc/

Nitric Oxide Nutritional remedies for hypertension and atherosclerosis. It’s 12 am: do you know where your electrons are?

Author and Reporter: Meg Baker, 10/7/2012.

http://pharmaceuticalintelligence.com/2012/10/07/no-nutritional-remedies-for-hypertension-and-atherosclerosis-its-12-am-do-you-know-where-your-electrons-are/

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Mitochondrial Damage and Repair under Oxidative Stress

Curator: Larry H Bernstein, MD, FCAP

 

Keywords: Mitochondria, mitochondrial dysfunction, electron transport chain, mtDNA, oxidative stress, oxidation-reduction, NO, DNA repair, lipid peroxidation, thiols, ROS, RNS, sulfur,base excision repair, ferredoxin.
Summary: The mitochondrion is the energy source for aerobic activity of the cell, but it also has regulatory functions that will be discussed. The mitochondrion has been discussed in other posts at this site. It has origins from organisms that emerged from an anaerobic environment, such as the bogs and marshes, and may be related to the chloroplast. The aerobic cell was an advance in evolutionary development, but despite the energetic advantage of using oxygen, the associated toxicity of oxygen abundance required adaptive changes. Most bacteria that reduce nitrate (producing nitrite, nitrous oxide or nitrogen) are called facultative anaerobes use electron acceptors such as ferric ions, sulfate or carbon dioxide which become reduced to ferrous ions, hydrogen sulfide and methane, respectively, during the oxidation of NADH (reduced nicotinamide adenine dinucleotide is a major electron carrier in the oxidation of fuel molecules).

The underlying problem we are left with is oxidation-reduction reactions that are necessary for catabolic and synthetic reactions, and that cumulatively damage the organism associated with cancer, cardiovascular disease, neurodegerative disease, and inflammatory overload. Aerobic organisms tolerate have evolved mechanisms to repair or remove damaged molecules or to prevent or deactivate the formationof toxic species that lead to oxidative stress and disease. However, the normal balance between production of pro-oxidant species and destruction by the antioxidant defenses is upset in favor of overproduction of the toxic species, which leads to oxidative stress and disease. How this all comes together is the topic of choice.

The transformation of energy is central to mitochondrial function. The system of energetics includes:

• the enzymes of the Kreb’s citric acid or TCA cycle,
• some of the enzymes involved in fatty acid catabolism (β-oxidation), and
• the proteins needed to help regulate these systems,

central to mitochondrial physiology through the production of reducing equivalents. Reducing equivalents are also used for anabolic reactions.
Electron Transport Chain
It also houses the protein complexes involved in the electron transport component of oxidative phosphorylation and proteins involved in substrate and ion transport. The chemical energy contained in both fats and amino acids can also be converted into NADH and FADH2 through mitochondrial pathways. The major mechanism for harvesting energy from fats is β-oxidation; the major mechanism for harvesting energy from amino acids and pyruvate is the TCA cycle. Once the chemical energy has been transformed into NADH and FADH2, these compounds are fed into the mitochondrial respiratory chain.
Under physiological conditions, electrons generally enter either through complex I (NADH-mediated, examined in vitro using substrates such as glutamate/malate) or complex II (FADH2-mediated, examined in vitro using succinate).

Electrons are then sequentially passed through a series of electron carriers.

The progressive transfer of electrons (and resultant proton pumping) converts the chemical energy stored in carbohydrates, lipids, and amino acids into potential energy in the form of the proton gradient. The potential energy stored in this gradient is used to phosphorylate ADP forming ATP.
Redox-Cycling

In redox cycling the reductant is continuously regenerated, thereby providing substrate for the “auto-oxidation” reaction.

When partially oxidized compounds are enzymatically reduced, the auto-oxidative generation of superoxide and other ROS to start again. Several enzymes

•  NADPH-cytochrome P450 reductase,
• NADPH-cytochrome b5 reductase [EC 1.6.2.2]
• NADPH-ubiquinone oxidoreductase [EC 1.6.5.3], and
• xanthine oxidase [EC 1.2.3.2]),

can reduce quinones into semiquinones in a single electron process.

The semiquinone can then reduce dioxygen to superoxide during its oxidation to a quinone.

Redox cycling is thought to play a role in carcinogenesis. The naturally occurring estrogen metabolites (the catecholestrogens) have been implicated in hormone-induced cancer, possibly as a result of their redox cycling and production of ROS. It is thought that diethylstilbestrol causes the production of the mutagenic lesion 8-hydroxy-2’deoxyguanosine. It can also cause DNA strand breakage.

Another oxidative reaction that is associated with H2O2 is a significant problem for living organisms as a consequence of the reaction between hydrogen peroxide and oxidizable metals, the Fenton reaction [originally described in the oxidation of an α-hydroxy acid to an α-keto acid in the presence of hydrogen peroxide (or hypochlorite) and low levels of iron salts (Fenton (1876, 1894)).
Chemical Reactions and Biological Significance

The hydroxyl free radical is so aggressive that it will react within 5 (or so) molecular diameters from its site of production. The damage caused by it, therefore, is very site specific. Biological defenses have evolved that reduce the chance that the hydroxyl free radical will be produced to repair damage. An antioxidant would have to occur at the site of hydroxyl free radical production and be at sufficient concentration to be effective.

Some endogenous markers have been proposed as a useful measures of total “oxidative stress” e.g., 8-hydroxy-2’deoxyguanosine in urine. The ideal scavenger

• must be non-toxic,
• have limited or no biological activity,
• readily reach the site of hydroxyl free radical production,
• react rapidly with the free radical, be specific for this radical, and
• neither the scavenger nor its product(s) should undergo further metabolism.

Unlike oxygen, nitrogen does not possess unpaired electrons and is therefore considered diamagnetic. Nitrogen does not possess available d orbitals so it is limited to a valency of 3. In the presence of oxygen, nitrogen can produce Nitric oxide which occurs physiologically with the immune system which, when activated, can produce large quantities of nitric oxide.

Nitric oxide is produced by stepwise oxidation of L-arginine catalyzed by nitric oxide synthase (NOS). Nitric oxide is formed from the guanidino nitrogen of the L-arginine in a reaction that

• consumes five electrons and
• requires flavin adenine dinucleotide (FAD),
• flavin mononucleotide (FMN) tetrahydrobiopterin (BH4), and
• iron protoporphyrin IX as cofactors.

The primary product of NOS activity may be the nitroxyl anion that is then converted to nitric oxide by electron acceptors.

NOS cDNAs show homology with the cytochrome P450 reductase family. Based on molecular genetics there appears to be at least three distinct forms of NOS:

• A Ca2+/calmodulin-requiring constitutive enzyme (c-NOS; ncNOS or type I)
• A calcium-independent inducible enzyme (i-NOS; type II), which is primarily involved in the mediation of the cellular immune response; and
• A second Ca2+/calmodulin-requiring constitutive enzyme found in aortic and umbilical endothelia (ec-NOS or type III)

This has been discussed extensively in this series of posts. Recently, a mitochondrial form of the enzyme, which appears to be similar to the endothelial form, has been found in brain and liver tissue. Although the exact role of nitric oxide in the mitochondrion remains elusive, it may play a role in the regulation of cytochrome oxidase.
Nitric Oxide
Nitric oxide appears to regulate its own production through a negative feedback loop. The binding of nitric oxide to the heme prosthetic group of NOS inhibits this enzyme, and c-NOS and ec-NOS are much more sensitive to this regulation than i-NOS. It appears that in the brain, NO can regulate its own synthesis and therefore the neurotransmission process.

• On the one hand, inhibition of ec-NOS will prevent the cytotoxicity associated with excessive nitric oxide production.
• On the other, the insensitivity of i-NOS to nitric oxide will enable high levels of nitric oxide to be produced for cytotoxic effects.

Endogenous inhibitors of NOS (guanidino-substituted derivatives of arginine) occur in vivo as a result of post-translational modification of protein contained arginine residues by S-adenosylmethionine. The dimethylarginines (NG,NG-dimethyl-L-arginine and NG,N’G-dimethyl-L-arginine) occurs in tissue proteins, plasma, and urine of humans and they are thought to act as both regulators of NOS activity and reservoirs of arginine for the synthesis of nitric oxide.
It has been calculated that even though membrane makes up about 3% of the total tissue volume, 90% of the reaction of nitric oxide with oxygen occurs within this compartment. Thus the membrane is an important site for nitric oxide chemistry.
There are two major aspects to nitric oxide chemistry.

• It can undergo single electron oxidation and reduction reactions producing nitrosonium and nitroxyl
• Having a single unpaired electron in its π*2p molecular orbital it will react readily with other molecules that also have unpaired electrons, such as free radicals and transition metals.

Examples of the reaction of nitric oxide with radical species include:

• Nitric oxide will react with oxygen to form the peroxynitrite (nitrosyldioxyl) radical (ONO2)
• and with superoxide to form the powerful oxidizing and nitrating agent, peroxynitrite anion (ONO2-). Peroxynitrite causes damage to many important biomolecules

Importance:

• nitrosothiols that are important in the regulation of blood pressure terminates lipid peroxidation
• 3-nitrosotyrosine and/or 4-O-nitrosotyrosine can affect the activity of enzymes that utilize tyrosyl radicals
• rapidly reacts with oxyhemoglobin, the primary route of its destruction in vivo
• the reaction between nitric oxide and transition metal complexes

During the last reaction a “ligand” bond is formed (the unpaired electron of nitric oxide is partially transferred to the metal cation),

 resulting in a nitrosated (nitrosylated) complex.

For example, such complexes can be formed with free iron ions,

iron bound to heme or iron located in iron-sulfur clusters.

Ligand formation allows nitric oxide to act as a signal, activating some enzymes while inhibiting others. Thus, the binding of nitric oxide to the Fe (II)-heme of guanylate (guanalyl) cyclase [GTP-pyrophosphate lyase: cyclizing] is the signal transduction mechanism. Guanylate cyclase exists as cytosolic and membrane-bound isozymes.
Thiol-Didulfide Redox Couple

The thiol-disulfide redox couple is very important to oxidative metabolism. For example, GSH is a reducing cofactor for glutathione peroxidase, an antioxidant enzyme responsible for the destruction of hydrogen peroxide.

The importance of the antioxidant role of the thiol-disulfide redox couple:

Thiols and disulfides can readily undergo exchange reactions, forming mixed disulfides. Thiol-disulfide exchange is biologically very important. For example,

• GSH can react with protein cystine groups and influence the correct folding of proteins.
• GSH may also play a direct role in cellular signaling through thiol-disulfide exchange reactions with membrane bound receptor proteins
•                        the insulin receptor complex)
•                        transcription factors (e.g., nuclear factor κB)
•                        and regulatory proteins in cells

Conditions that alter the redox status of the cell can have important consequences on cellular function.

The generation of ROS by redox cycling is only one possible explanation for the action of many drugs. Rifamycin not only owes its activity to ROS generation but also to its ability to block bacterial RNA synthesis as well. Quinones (and/or semiquinones) can also form adducts with nucleophiles, especially thiols. These adducts may act as toxins directly or indirectly through the inhibition of key enzymes (e.g., by reacting with essential cysteinyl residues) or the depletion of GSH.
DNA Adduct Formation

By far the most intense research in this field has been directed towards the chemistry and biology of DNA adduct formation. Attack of the free bases and nucleosides by pro-oxidants can yield a wide variety of adducts and DNA-protein cross-links. Such attack usually occurs

• at the C-4 and C-8 position of purines and
• C-5 and C-6 of pyrimidines.

Hydroxyl free radical-induced damage to purine bases and nucleosides can proceed through a C-8-hydroxy N-7 radical intermediate, and then either undergo oxidation with the production of an 8-hydroxy purine, or reduction, probably by cellular thiols, followed by ring opening and the formation of FAPy (formamido-pyrimidine) metabolites (hydroxyl free radical-induced damage to guanosine). Although most research has focused on 8-hydroxy-purine adducts a growing number of publications are attempting to measure the FAPy derivative.

Nitrosation of the Amines of the Nucleic Acid Bases.

Primary aromatic amines produce deaminated products, while secondary amines form N-nitroso compounds.
Formation of Peroxynitrite from Nitric Oxide.

Peroxynitrite shows complex reactivity

• with DNA initiating DNA strand breakage, oxidation (e.g., formation of 8-hydroxyguanine, 8-OH2’dG, (5-hydroxymethyl)-uracil, and FAPyGua),
• nitration (e.g., 8-nitroguanine), and
• deamination of bases.

Peroxynitrite can also promote the production of lipid peroxidation related active carbonyls and cause the activation of NAD+ ADP-ribosyltransferase.

Modification of Guanine
Although all DNA bases can be oxidatively damaged, it is the modification of guanine that is the most frequent. 8OH2’dG is the most abundant DNA adduct. This can affect its hydrogen bonding between base-pairs. These base-pair substitutions are usually found clustered into areas called “hot spots”. Guanine normally binds to cytosine.

8OH2’dG, however, can form hydrogen bonds with adenine. The formation of 8OH2’dG in DNA can therefore result in a G→T transversion.

8-Hydroxyguanine was also shown to induce codon 12 activation of c-Ha-ras and K-ras in mammalian systems. G→T transversions are also the most frequent hot spot mutations found in the p53 supressor gene which is associated with human tumors.

Other mechanisms by which ROS/RNS can lead to mutations have been
proposed. Direct mechanisms include:

• conformational changes in the DNA template that reduces the accuracy of replication by DNA polymerases
• altered methylation of cytosine that affects gene control

Indirect mechanisms include:

• Oxidative damage to proteins, including DNA polymerases and repair enzymes.
• Damage to lipids causes the production of mutagenic carbonyl compounds
• Misalignment mutagenesis (“slippery DNA”)

DNA Mismatch Repair 5 (Photo credit: Allen Gathman)

Repair of ROS/RNS-induced DNA Damage
The repair of damaged DNA is an ongoing and continuous process involving a
number of repair enzymes. Damaged DNA appears to be mended by two major mechanisms:

1. base excision repair (BER) and
2. nucleotide excision repair (NER)

Isolated DNA is found to contain low levels of damaged bases, so it appears that these repair processes are not completely effective.
Base Excision Repair

BER is first started by DNA glycosylases which recognize specific base
modifications (e.g., 8OH2’dG). For example,

• Formamido-pyrimidine-DNA glycosylase (Fpg protein) recognizes damaged purines such as 8-oxoguanine and FAPyGua.
• Damaged pyrimidines are recognized by endonuclease III, which acts as both a glycosylase and AP endonuclease.
• Glycosylases cleave the N-glycosylic bond between the damaged base and the sugar

Following the glycosylase step, AP endonucleases then remove the 3′-deoxyribose moiety by cleavage of the phosphodiester bonds thereby generating a 3’-hydroxyl group that can then be extended by DNA polymerase.

The final step in mending damaged DNA is the rejoining of the free ends of DNA by a DNA ligase. It also appears that the presence of 8-oxoguanine modified bases in DNA is not only a result of ROS attack on this macromolecule. Oxidized nucleosides and nucleotides from free cellular pools can also be incorporated into DNA by polymerases and cause AT to CG base substitution mutations.

Mitochondrial DNA Repair

The mitochondrion genome encodes the various complexes of the electron transport chain, but contains no genetic information for DNA repair enzymes. These enzymes must be obtained from the nucleus. As mitochondria are continuously producing DNA damaging pro-oxidant species, effective DNA repair mechanisms must exist within the mitochondrial matrix in order for these organelles to function. Mitochondria have a short existence, and excessively damaged mitochondria will be quickly removed. Mitochondria contain many BER enzymes and are proficient at repair, but they do not appear to repair damaged DNA by NER mechanisms.

Single Strand DNA Damage and PARP Activation

Single strand DNA breakage activates NAD+ ADP-ribosyltransferase (PARP). PARP is a protein-modifying, nucleotide-polymerizing enzyme and is found at high levels in the nucleus. Activated PARP

1. cleaves NAD+ into ADP-ribose and nicotinamide
2. then attaches the ADP-ribose units to a variety of nuclear proteins (including histones and its own automodification domain).
3. then polymerizes the initial ADP-ribose modification with other ADP-ribose units to form the nucleic acid-like polymer, poly (ADP) ribose.

PARP only appears to be involved with BER and not NER. In BER PARP does not appear to play a direct role but rather it probably helps by keeping the chromatin in a conformation that enables other repair enzymes to be effective. It may also provide temporary protection to DNA molecules while it is being repaired. Conflicting evidence suggests that PARP may not be an important DNA repair enzyme as cells from a PARP knockout mouse model have normal repair characteristics.

Activation of PARP can be dangerous to the cell. For each mole of ADP-ribose transferred, one mole of NAD+ is consumed, and through the regeneration of NAD+ four ATP molecules are wasted. Thus the activation of PARP can rapidly deplete a cell’s energy store and even lead to cell death. Some researchers suggest that this may be one mechanism whereby cells with excessive DNA damage are effectively removed. However, a variety of diseases may involve PARP overactivation including

• circulatory shock,
• CNS injury,
• diabetes,
• drug-induced cytotoxicity, and
• inflammation.

The Indirect Pathway.
This (mutation) pathway does not involve oxidative damage to the protein per se. This process involves oxidative damage to the DNA molecule encoding the protein. Thus pro-oxidants can cause changes in the base sequence of the DNA molecule. If such base modification is in a coding region of DNA (exon) and not corrected, the DNA molecule may be transcribed incorrectly. Translation of the mutant mRNA can result in a mutant protein containing a wrong amino acid in its primary sequence. If this modified amino acid occurs in an essential part of the protein (e.g., the active site of an enzyme or a portion that alters folding), the function of that protein may be impaired. Fortunately, unlike modified DNA
that can pass from cell to cell during mitosis thereby continuing the production of mutant protein, damage to a protein is non-replicating and stops with its destruction.

The Direct Pathway

This (post-translational) pathway involves the action of a pro-oxidant on a protein resulting in

• modification of amino acid residues,
• the formation of carbonyl adducts,
• cross-linking and
• polypeptide chain fragmentation.

Such changes often result in altered protein conformation and/or activity. Proteins will produce a variety of carbonyl products when exposed to metal-based systems (metal/ascorbate and metal/hydrogen peroxide) in vitro. For example, histidine yields aspartate, asparagine and 2-oxoimidazoline, while proline produces glutamate, pyroglutamate, 4-hydroxyproline isomers, 2-pyrrolidone and γ-aminobutyric acid. Metal-based systems and other pro-oxidant conditions can oxidize methionine to its sulfoxide.

This portion of the presentation is endebted to THE HANDBOOK OF REDOX
BIOCHEMISTRY, Ian N. Acworth, August 2003, esa. (inacworth@esainc.com).
We shall now identify more recent work related to this presentation.

Oxygen and Oxidative Stress

The reduction of oxygen to water proceeds via one electron at a time. In the mitochondrial respiratory chain, Complex IV (cytochrome oxidase) retains all partially reduced intermediates until full reduction is achieved. Other redox centres in the electron transport chain, however, may leak electrons to oxygen, partially reducing this molecule to superoxide anion (O2_•). Even though O2_• is not a strong oxidant, it is a precursor of most other reactive oxygen species, and it also becomes involved in the propagation of oxidative chain reactions. Despite the presence of various antioxidant defences, the mitochondrion appears to be the main intracellular source of these oxidants. This review describes the main mitochondrial sources of reactive species and the antioxidant defences that evolved to prevent oxidative damage in all the mitochondrial compartments.

Reactive oxygen species (ROS) is a phrase used to describe a variety of molecules and free radicals (chemical species with one unpaired electron) derived from molecular oxygen. Molecular oxygen in the ground state is a bi-radical, containing two unpaired electrons in the outer shell (also known as a triplet state).

Since the two single electrons have the same spin, oxygen can only react with one electron at a time and therefore it is not very reactive with the two electrons in a chemical bond.

On the other hand, if one of the two unpaired electrons is excited and changes its spin, the resulting species (known as singlet oxygen) becomes a powerful oxidant as the two electrons with opposing spins can quickly react with other pairs of electrons, especially double bonds.

The formation of OH• is catalysed by reduced transition metals, which in turn may be re-reduced by O2 -•, propagating this process. In addition, O2-• may react with other radicals including nitric oxide (NO•) in a reaction controlled by the rate of diffusion of both radicals. The product, peroxynitrite, is also a very powerful oxidant. The oxidants derived from NO• have been recently called reactive nitrogen species (RNS).

‘Oxidative stress’ is an expression used to describe various deleterious processes resulting from an imbalance between the excessive formation of ROS and/or RNS and limited antioxidant defences.

• Whilst small fluctuations in the steady-state concentration of these oxidants may actually play a role in intracellular signalling,
• uncontrolled increases in the steady-state concentrations of these oxidants lead to free radical mediated chain reactions

which indiscriminately target

• proteins,
• lipids,
• polysaccharides.

In vivo, O2-• is produced both enzymatically and nonenzymatically.

Enzymatic sources include

• NADPH oxidases located on the cell membrane of
• polymorphonuclear cells,
• macrophages and
• endothelial cells and
• cytochrome P450-dependent oxygenases.

The proteolytic conversion of xanthine dehydrogenase to xanthine oxidase provides another enzymatic source of both O2 -• and H2O2 (and therefore constitutes a source of OH•) and has been proposed to mediate deleterious processes in vivo.

Given the highly reducing intramitochondrial environment, various respiratory components, including flavoproteins, iron–sulfur clusters and ubisemiquinone, are thermodynamically capable of transferring one electron to oxygen. Moreover, most steps in the respiratory chain involve single-electron reactions, further favouring the monovalent reduction of oxygen. On the other hand, the mitochondrion possesses various antioxidant defences designed to eliminate both O2- • and H2O2.

The rate of O2 -• formation by the respiratory chain is controlled primarily by mass action, increasing both when electron flow slows down (increasing the concentration of electron donors, R•) and when the concentration of oxygen increases (eqn (1); Turrens et al. 1982).

d[O2]/dt = k [O2] [R•].

The energy released as electrons flow through the respiratory chain is converted into a H+ gradient through the inner mitochondrial membrane (Mitchell, 1977). This gradient, in turn, dissipates through the ATP synthase complex (Complex V) and is responsible for the turning of a rotor-like protein complex required for ATP synthesis. In the absence of ADP,

• the movement of H+ through ATP synthase ceases and
• the H+ gradient builds up
• causing electron flow to slow down and
• the respiratory chain to become more reduced (State IV respiration).

Mitochondrial Antioxidant Defences

The deleterious effects resulting from the formation of ROS in the mitochondrion are, to a large extent, prevented by various antioxidant systems. Superoxide is enzymatically converted to H2O2 by a family of metalloenzymes called superoxide dismutases (SOD). Since O2-• may either reduce transition metals, which in turn can react with H2O2 producing OH• or spontaneously react with NO• to produce peroxynitrite, it is important to maintain the steady-state concentration of O2-• at the lowest possible level. Thus, although the dismutation of O2-• to H2O2 and O2 can also occur spontaneously, the role of SODs is to increase the rate of the reaction to that of a diffusion-controlled process.

The mitochondrial matrix contains a specific form of SOD, with manganese in the active site, which eliminates the O2 -• formed in the matrix or on the inner side of the inner membrane. The expression of MnSOD is further induced by agents that cause oxidative stress, including radiation and hyperoxia, in a process mediated by the oxidative activation of the nuclear transcription factor NFkB .

Turrens JF. Mitochondrial formation of reactive oxygen species. J Physiol 2003; 552(2): 335–344. DOI: 10.1113/jphysiol.2003.049478. http://www.jphysiol.org

Reactive Oxygen Species and Control of Apoptosis

Reactive oxygen species (ROS) are products of normal metabolism and xenobiotic exposure, and depending on their concentration, ROS can be beneficial or harmful to cells and tissues.

• At physiological low levels, ROS function as “redox messengers” in intracellular signaling and regulation, whereas
• excess ROS induce oxidative modification of cellular macromolecules, inhibit protein function, and promote cell death.

Additionally, various redox systems, such as

• the glutathione,
• thioredoxin, and
• pyridine nucleotide redox couples,
• NADPH and antioxidant defense
• NAD+ and the function of sirtuin proteins

participate in cell signaling and modulation of cell function, including apoptotic cell death. Cell apoptosis is initiated by extracellular and intracellular signals via two main pathways,

1. the death receptor and
2. the mitochondria-mediated pathways.

ROS and JNK-mediated apoptotic signaling

              GSH redox status and apoptotic signaling

Various pathologies can result from oxidative stress-induced apoptotic signaling that is consequent to

• ROS increases and/or antioxidant decreases,
• disruption of intracellular redox homeostasis, and
• irreversible oxidative modifications of lipid, protein, or DNA.

We focus on several key aspects of ROS and redox mechanisms in apoptotic signaling and highlight the gaps in knowledge and potential avenues for further investigation. A full understanding of the redox control of apoptotic initiation and execution could underpin the development of therapeutic interventions targeted at oxidative stress-associated disorders.

Circu, M. L.; Aw, T. Y., Reactive oxygen species, cellular redox systems, and apoptosis, Free Radic. Biol. Med. 2010. FRB-10057; pp 14. doi:10.1016/j.freeradbiomed.2009.12.022

Assembly of Iron-sulfur (FeyS) Clusters

Iron-sulfur (FeyS) cluster-containing proteins catalyze a number of electron transfer and metabolic reactions. The components and molecular mechanisms involved in the assembly of the FeyS clusters have been identified only partially. In eukaryotes, mitochondria have been proposed to execute a crucial task in the generation of intramitochondrial and extramitochondrial FeyS proteins. Herein, we identify the essential ferredoxin Yah1p of Saccharomyces cerevisiae mitochondria as a central component of the FeyS protein biosynthesis machinery. Depletion of Yah1p by regulated gene expression resulted in a

30-fold accumulation of iron within mitochondria,

similar to what has been reported for other components involved in FeyS protein biogenesis. Yah1p was shown to be required for the assembly of FeyS proteins both inside mitochondria and in the cytosol. Apparently, at least one of the steps of FeyS cluster biogenesis within mitochondria requires reduction by ferredoxin. Our findings lend support to the idea of a primary function of mitochondria in the biosynthesis of FeyS proteins outside the organelle. To our knowledge, Yah1p is the first member of the ferredoxin family for which a function in FeyS cluster formation has been established. A similar role may be predicted for the bacterial homologs that are encoded within iron-sulfur cluster assembly (isc) operons of prokaryotes.
H Lange, A Kaut, G Kispal, and R Lill. A mitochondrial ferredoxin is essential for biogenesis of cellular iron-sulfur proteins. PNAS 2000; 97(3): 1050–1055.

DNA Charge Transport

Damaged bases in DNA are known to lead to errors in replication and transcription, compromising the integrity of the genome. The authors proposed a model where repair proteins containing redoxactive [4Fe-4S] clusters utilize DNA charge transport (CT) as a first step in finding lesions. In this model, the population of sites to search is reduced by a localization of protein in the vicinity of lesions. Here, we examine this model using single-molecule atomic force microscopy (AFM). XPD, a 5′-3′ helicase involved in nucleotide
excision repair, contains a [4Fe-4S] cluster and exhibits a DNA bound redox potential that is physiologically relevant.

In AFM studies, they observe the redistribution of XPD onto kilobase DNA strands containing a single base mismatch, which is not a specific substrate for XPD but, like a lesion, inhibits CT. They also provide evidence for DNA-mediated signaling between XPD and Endonuclease III (EndoIII), a base excision repair glycosylase that also contains a [4Fe-4S] cluster.

• When XPD and EndoIII are mixed together, they coordinate in relocalizing onto the mismatched strand.
• However, when a CT-deficient mutant of either repair protein is combined with the CT-proficient repair partner, no relocalization occurs.

The data presented here indicate that XPD, an archaeal protein from the NER pathway, may cooperate with other proteins that are proficient at DNA CT to localize in the vicinity of damage. XPD, a superfamily 2 DNA helicase with 5′-3′ polarity, is a component of TFIIH that is essential for repair of bulky lesions generated by exogenous sources such as UV light and chemical carcinogens. XPD contains a conserved [4Fe-4S] cluster suggested to be conformationally controlled by ATP binding and hydrolysis.

Mutations in the iron-sulfur domain of XPD can lead to diseases including TTD and XP, yet the function of the [4Fe-4S] cluster appears to be unknown.

Electrochemical studies have shown that when BER proteins MutY and EndoIII bind to DNA, their [4Fe-4S] clusters are activated toward one electron oxidation. XPD exhibits a DNA-bound midpoint potential similar to that of EndoIII and MutY when bound to DNA (approximately 80 mV vs. NHE), indicative of a possible role for the [4Fe-4S] cluster in DNA-mediated CT.

For EndoIII we have also already determined a direct correlation between the ability of proteins to redistribute in the vicinity of mismatches as measured by AFM, and the CT proficiency of the proteins measured electrochemically. Thus, we may utilize single-molecule AFM as a tool to probe the redistribution of proteins in the vicinity of base lesions and in so doing, the proficiency of the protein to carry out DNA CT.

Here we show that, like the BER protein EndoIII, XPD, involved both in transcription and NER, redistributes in the vicinity of a lesion. Importantly, this ability to relocalize is associated with the ability of XPD to carry out DNA CT. The mutant L325V is defective in its ability to carry out DNA CTand this XPD mutant also does not redistribute effectively onto the mismatched strand.

These data not only indicate a general link between the ability of a repair protein to carry out DNA CT and its ability to redistribute onto DNA strands near lesions but also provide evidence for coordinated DNA CT between different repair proteins in their search for damage in the genome. These data also provide evidence that two different repair proteins, each containing a [4Fe-4S] cluster at similar DNA bound potential, can communicate with one another through DNA-mediated CT.

Sontz PA, Mui TP, Fuss JO, Tainer JA, and Barton JK. DNA charge transport as a first step in coordinating the detection of lesions by repair proteins. PNAS 2012; 109(6):1856–1861. doi:10.1073/pnas.1120063109/-/ DCSupplemental. http://www.pnas.org/lookup/suppl/

Janus Bifron 

The signaling function of mitochondria is considered with a special emphasis on their role in the regulation of redox status of the cell, possibly determining a number of pathologies including cancer and aging. The review summarizes the transport role of mitochondria in energy supply to all cellular compartments (mitochondria as an electric cable in the cell), the role of mitochondria in plastic metabolism of the cell including synthesis of

• heme,
• steroids,
• iron-sulfur clusters, and
• reactive oxygen and nitrogen species.

Mitochondria also play an important role in the Ca2+-signaling and the regulation of apoptotic cell death. Knowledge of mechanisms responsible for apoptotic cell death is important for the strategy for prevention of unwanted degradation of postmitotic cells such as cardiomyocytes and neurons.

In accordance with P. Mitchell’s chemiosmotic concept, vectorial transmembrane transfer of electrons and protons is accompanied by generation of electrochemical difference of proton electrochemical potential on the inner mitochondrial membrane; its utilization by ATP synthase induces conformational rearrangements resulting in ATP synthesis from ADP and inorganic phosphate. Details of the mechanism responsible for ATP synthesis are given elsewhere.

Membrane potential (DY) generated across the inner mitochondrial membrane is the component of the transmembrane electrochemical potential of H+ ions (DμH+), which provides ATP synthesis together with the concentration component (DpH). Maintenance of constant membrane potential is a vitally important precondition for functioning of mitochondria and the cell. Under conditions of limited supply of the cell with oxygen (hypoxia) and inability to carry out aerobic ATP synthesis, mitochondria become ATP consumers (rather than generators) and ATP is hydrolyzed by mitochondrial ATPase, and this is accompanied by generation of membrane potential.

Redox homeostasis, i.e. the sum of redox components (including proteins, low molecular weight redox components such as NAD/NADH, flavins, coenzymes Q, oxidized and reduced substrates, etc.) is one of important preconditions for normal cell functioning.

Single-strand and double-strand DNA damage (Photo credit: Wikipedia)

Mitochondria generate such potent regulators of redox potential as

• superoxide anion,
• hydrogen peroxide,
• nitric oxide,
• peroxynitrite, etc.

They are actively involved in regulation of cell redox potential and consequently

• control proteolysis,
• activation of transcription,
• changes in mitochondrial DNA (mDNA),
• cell metabolism, and
• cell differentiation.

Zorov DB, Isaev NK, Plotnikov EY, Zorova LD, et al. The Mitochondrion as Janus Bifrons. Biochemistry (Moscow) 2007; 72(10): 1115-1126. ISSN 0006-2979.
DOI: 10.1134/S0006297907100094

Structure of the human mitochondrial genome. (Photo credit: Wikipedia)

Gene Expression Associated with Oxidoreduction and Mitochondria
The naked mole-rat (Heterocephalus glaber) is a long-lived, cancer resistant rodent and there is a great interest in identifying the adaptations responsible for these and other of its unique traits. We employed RNA sequencing to compare liver gene expression profiles between naked mole-rats and wild-derived mice. Our results indicate that genes associated with oxidoreduction and mitochondria were expressed at higher relative levels in naked mole-rats. The largest effect is nearly

300-fold higher expression of epithelial cell adhesion molecule (Epcam), a tumour-associated protein.

Also of interest are the

• protease inhibitor, alpha2-macroglobulin (A2m), and the
• mitochondrial complex II subunit Sdhc,

both ageing-related genes found strongly over-expressed in the naked mole-rat.

These results hint at possible candidates for specifying species differences in ageing and cancer, and in particular suggest complex alterations in mitochondrial and oxidation reduction pathways in the naked mole-rat. Our differential gene expression analysis obviated the need for a reference naked mole-rat genome by employing a combination of Illumina/Solexa and 454 platforms for transcriptome sequencing and assembling transcriptome contigs of the non-sequenced species. Overall, our work provides new research foci and methods for studying the naked mole-rat’s fascinating characteristics.

C Yu, Y Li, A Holmes, K Szafranski, CG Faulkes, et al. RNA Sequencing Reveals Differential Expression of Mitochondrial and Oxidation reduction Genes in the Long-Lived Naked Mole-Rat When Compared to Mice. PLoS ONE 2011; 6(11): 1-9. e26729. http://www.plosone.org

The complete set of viable deletion strains in Saccharomyces cerevisiae was screened for sensitivity of mutants to five oxidants to identify cell functions involved in resistance to oxidative stress. This screen identified a unique set of mainly constitutive functions providing the first line of defense against a particular oxidant; these functions are very dependent on the nature of the oxidant. Most of these functions are distinct from those involved in repair and recovery from damage, which are generally induced in response to stress, because there was little correlation between mutant sensitivity and
the reported transcriptional response to oxidants of the relevant gene. The screen identified 456 mutants sensitive to at least one of five different types of oxidant, and these were ranked in order of sensitivity. Many genes identified were not previously known to have a role in resistance to reactive oxygen species. These encode functions including

• protein sorting,
• ergosterol metabolism,
• autophagy, and
• vacuolar acidification.

two mutants were sensitive to all oxidants examined,
12 were sensitive to at least four,

Different oxidants had very different spectra of deletants that were sensitive. These findings highlight the specificity of cellular responses to different oxidants:

• No single oxidant is representative of general oxidative stress.
• Mitochondrial respiratory functions were overrepresented in mutants sensitive to H2O2, and
• vacuolar protein-sorting mutants were enriched in mutants sensitive to diamide.

Core functions required for a broad range of oxidative-stress resistance include

• transcription,
• protein trafficking, and
• vacuolar function.

GW Thorpe, CS Fong, N Alic, VJ Higgins, and IW Dawes. Cells have distinct mechanisms to maintain protection against different reactive oxygen species: Oxidative-stress-response genes. PNAS 2004;101: 6564–6569. http://www.pnas.org cgi doi 10.1073 pnas.0305888101
Subcellular Thiol Redox State in Complex I Deficiency

Isolated complex I deficiency is the most common enzymatic defect of the oxidative phosphorylation (OXPHOS) system, causing a wide range of clinical phenotypes. Th authers reported before that the rates at which reactive oxygen species (ROS)-sensitive dyes are converted into their fluorescent oxidation products are markedly increased in cultured skin fibroblasts of patients with nuclear-inherited isolated complex I deficiency.

Using videoimaging microscopy we show here that these cells also display a marked increase in NAD(P)H autofluorescence. Linear regression analysis revealed a negative correlation with the residual complex I activity and a positive correlation with the oxidation rates of the ROS sensitive dyes (5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein and hydroethidine for a large cohort of 10 patient cell lines.

On the other hand, video-imaging microscopy of cells selectively expressing reduction-oxidation sensitive GFP1 in either the mitochondrial matrix or cytosol showed the absence of any detectable change in thiol redox state. In agreement with this result, neither the glutathione nor the glutathione disulfide content differed significantly between patient and healthy fibroblasts.

Finally, video-rate confocal microscopy of cells loaded with C11-BODIPY581/591 demonstrated that the extent of lipid peroxidation, which is regarded as a measure of oxidative damage, was not altered in patient fibroblasts. Our results indicate that fibroblasts of patients with isolated complex I deficiency maintain their thiol redox state despite marked increases in ROS production.

S Verkaart, WJH Koopman, J Cheek, SE van Emst-de Vries. Mitochondrial and cytosolic thiol redox state are not detectably altered in isolated human NADH:ubiquinone oxidoreductase deficiency. Biochimica et Biophysica Acta (BBA) – Molecular Basis of Disease 2007; 1772(9): 1041. DOI : 10.1016/j.bbadis.2007.05.004

• Mitochodrial mtDNA and Cancer
• Mitochondrial research has recently been driven by the

identification of mitochondria-associated diseases and 
the role of mitochondria in apoptosis.

Moreover, mitochondria have been implicated in the process of carcinogenesis because of their vital role in

• energy production,
• nuclear-cytoplasmic signal integration and
• control of metabolic pathways.

At some point during neoplastic transformation, there is an increase in reactive oxygen species (ROS), which damage the mitochondrial genome. This accelerates the somatic mutation rate of mitochondrial DNA.

Mitochondrial characteristics

There are several biological characteristics which cast mitochondria and, in particular, the mitochondrial genome, as a biological tool for early detection and monitoring of neoplasia and its potential progression. These vital characteristics are important in cancer research, as not all neoplasias become malignant. Mitochondria are archived in the cytoplasm of the ovum and as such do not recombine.

This genome has an accelerated mutation rate, by comparison with the nucleus, and accrues somatic mutations in tumour tissue. Moreover, mitochondrial DNA (mtDNA) has a high copy number in comparison with the nuclear archive of DNA. There are potentially thousands of mitochondrial genomes per cell, which enables detection of important biomarkers, even at low levels. In addition, mtDNA can be heteroplasmic, which means that disease-associated mutations occur in a subset of the genomes.

The presence of heteroplasmy is an indication of disease and is found in many human tumours. Identification of low levels of heteroplasmy may allow unprecedented early identification and monitoring of neoplastic progression to malignancy.

Coding for just 13 enzyme complex subunits, 22 transfer RNAs and two ribosomal RNAs, the mitochondrial genome is packaged in a compact 16,569 base pair (bp) circular molecule. These products participate in the critical electron transport process of ATP production. Collectively, mitochondria generate 80 per cent of the chemical fuel which fires cellular metabolism.

As a result, nuclear investment in the mitochondria is high — that is, several thousand nuclear genes control this organelle in order to accomplish the complex interactions required to maintain a network of pathways, which coordinate energy demand and supply.

It has been proposed that these mutations may serve as an early indication of potential cancer development and may represent a means for tracking tumour progression.

Does this provide a potential utility in that these mutations may be used for the identification and monitoring of neoplasia and malignant transformation where appropriate body fluids or non-invasive tissue access is available for mtDNA recovery? Specifically discussed are:

• prostate,
• breast,
• colorectal,
• skin and
• lung cancers

There are many important questions yet to be addressed: such as

• the relationship between mtDNA and the actual disease;
• are mutations causative or merely a reflection of nuclear instability?
• And, are these processes independent events?

Alterations in the non-coding D-loop suggest genome instability;
however, as studies focus more on the coding regions of the
mitochondrial genome,

Particularly in the case of nonsynonymous mutations in the genes
contributing products to the electron transport process, metabolic
implications are evident. Moreover, mutations in mitochondrial
transfer RNAs indicate the possibility of a global mitochondrial
translational shut down.

RL Parr, GD Dakubo, RE Thayer, K McKenney, MA Birch-Machin. Mitochondrial DNA as a potential tool for early cancer detection. HUMAN GENOMICS 2006; 2(4). 252–257.
Mitochondrial DNA (mtDNA) is particularly prone to oxidation due to the lack of histones and a deficient mismatch repair system. This explains an increased mutation rate of mtDNA that results in heteroplasmy, e.g., the coexistence of the mutant and wild-type mtDNA molecules within the same mitochondrion. Hyperglycemia is a key risk factor not only for diabetes-related disease, but also for cardiovascular and all-cause mortality. One can assume an increase in the risk of cardiovascular disease by 18% for each unit (%) glycated hemoglobin HbA1c. In the Glucose Tolerance in Acute Myocardial Infarction study of patients with acute coronary syndrome, abnormal glucose tolerance was the strongest independent predictor of subsequent cardiovascular complications and death. In the Asian Pacific Study, fasting plasma glucose was shown to be an independent predictor of cardiovascular events up to a level of 5.2 mmol/L.

Glucose level fluctuations and hyperglycemia are triggers for inflammatory responses via increased mitochondrial superoxide production and endoplasmic reticulum stress. Inflammation leads to insulin resistance and β-cell dysfunction, which further aggravates hyperglycemia. The molecular pathways that integrate hyperglycemia, oxidative stress, and diabetic vascular complications have been most clearly described in the pathogenesis of endothelial dysfunction, which is considered as the first step in atherogenesis according to the response to injury hypothesis.

• In diabetes mellitus,
• glycotoxicity,
• advanced oxidative stress,
• collagen cross-linking, and
• accumulation of lipid peroxides

in foam macrophage cells and arterial wall cells may significantly

• decrease the mutation threshold,
• endothelial dysfunction,
• promoting atherosclerosis.

Alterations in mitochondrial DNA (mtDNA), known as homoplasmic and heteroplasmic mutations, may influence mitochondrial OXPHOS capacity, and in turn contribute to the magnitude of oxidative stress in micro- and macrovascular networks in diabetic patients.
The authors critically consider the impact of mtDNA mutations on the pathogenesis of cardiovascular diabetic complications.

Mutation Threshhold

Although cells may harbor mutant mtDNA, the expression of disease is dependent on the percent of alleles bearing mutations. Modeling confirms that an upper threshold level might exist for mutations beyond which the mitochondrial population collapses, with a subsequent decrease in ATP. This decrease in ATP results in the phenotypic expression of disease. It is estimated that in many patients with clinical manifestations of mitochondrial disorders, the proportion of mutant DNA exceeds 50%.

For the MELAS (mitochondrial encephalopathy, lactic acidosis and stroke-like syndrome)-causing mutation m.3243 A>G in the mitochondrial gene encoding tRNALeu, which is also associated with diabetes plus deafness, a strong correlation between the level of mutational heteroplasmy and documented disease has been found. Increased percentages of mutant mtDNA in muscle cells (up to 71%) can lead to mitochondrial myopathy. Levels of heteroplasmy of over 80% may lead to recurrent stroke and mutation levels of 95% have been associated with MELAS.

Regardless of the type of mutation or the level of heteroplasmy in affected mitochondria, unrepaired damage leads to a decrease in ATP, which in turn causes the phenotypic manifestation of disease. The manifestation of disease not only depends on the ATP level but also on the tissue affected. Various tissues have differing levels of demand on OXPHOS capacity. To evaluate a tissue threshold, Leber’s hereditary optic neuropathy can be used as a model for mitochondrial neurodegenerative disease. For neural and skeletal muscle tissues, the tissue threshold should be as high as or higher than 90% of
damaged (mutated) mtDNA. To induce mitochondrial malfunctions, the tissue threshold of the cardiac muscle is estimated to be significantly lower (approximately 64%-67%). In chronic vascular disease such as atherosclerosis, a mutation threshold in the affected vessel wall (e.g., in the postmortem aortic atherosclerotic plaques) was observed to be significantly lower. For example, for mutations m.3256 C>T, m.12315 G>A, m.15059 G>A, and m.15315 G>A, the heteroplasmy range of 18%-66% in the atherosclerotic lesions was 2-3.5-fold that in normal vascular tissue.

Mitochondrial stress and insulin resistance

• Mitochondrial damage precedes the development of atherosclerosis and tracks the extent of the lesion in apoE-null mice, and
• mitochondrial dysfunction caused by heterozygous deficiency of a superoxide dismutase increases atherosclerosis and vascular mitochondrial damage in the same model.

Blood vessels destined to develop atherosclerosis may be characterized by inefficient ATP production due to the uncoupling of respiration and OXPHOS. Blood vessels have regions of hypoxia, which lower the ratio of state 3 (phosphorylating) to state 4 (nonphosphorylating) respiration. Human atherosclerotic lesions have been known for decades to be deficient in essential fatty acids, a condition that causes respiratory uncoupling and atherosclerosis.

The finding by Kokaze et al.  helps to explain, at least in part, the anti-atherogenic effect of the allele m. 5178A due to its relation with the favorable lipid profile. The nucleotide change causes leucine-to-methionine substitution at codon 237 (Leu-237Met) of the NADH dehydrogenase subunit 2 located in the loop between 7th and 8th transmembrane domains of the mitochondrial protein. Given that this methionine residue is exposed at the surface of respiratory Complex I, this residue may be available as an efficient oxidant scavenger. Complex I

• accepts electrons from NADH,
• transfers them to ubiquinone, and
• uses the energy released to pump protons across the mitochondrial inner membrane.

Thus, the Leu237Met replacement in the ND2 subunit might have a protective effect against oxidative damage to mitochondria.

Most fatty acid oxidation, which is promoted by peroxisome proliferator-activated receptor α (PPARα) activation, occurs in the mitochondria. Mitochondrial effects could explain why PPARα- deficient mice are protected from diet-induced insulin resistance and atherosclerosis as well as glucocorticoid induced insulin resistance and hypertension. Caloric restriction,

• improves features of insulin resistance,
• increases mitochondrial biogenesis and, surprisingly,
• enhances the efficiency of ATP production.

Dysfunctional mitochondria in cultured cells can be rescued by transfer of mitochondria from adult stem cells, raising the possibility of restoration of normal bioenergetics in the vasculature to treat atherosclerosis associated with insulin resistance.
Chistiakov DA, Sobenin IA, Bobryshev YV, Orekhov AN. Mitochondrial dysfunction and mitochondrial DNA mutations in atherosclerotic complications in diabetes. World J Cardiol 2012; 4(5): 148-156. ISSN 1949-8462 (online). doi:10.4330/wjc.v4.i5.148. http://www.wjgnet.com/1949-8462/full/v4/i5/148.htm

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Nitric Oxide and Sepsis, Hemodynamic Collapse, and the Search for Therapeutic Options

dimethyl arginineNitric Oxide and Sepsis

Curator, Reporter, EAW: Larry H Bernstein, MD, FCAP

This document explores the current understanding of sepsis as a cascade of events that involves the microcirculation unevenly because of a differential effect on the large and contiguous intestinal epithelium, secondary effects on cardiopulmonary blood flows and cardiac output, and the role of Nitric Oxide in the emergence of beneficial and potentially deleterious effects. This leads to a substantial body of work on therapeutic targets, either aimed at total inhibition or selective inhibition of NO synthase, and the special role of iNOS. This is another of a series of discussions on the metabolic and regulatory role of NO in health and disease.

Introduction

Antioxidants are essential, and are involved in several important biological processes such as immunity, protection against tissue damage, reproduction, growth and development. Antioxidants preserve adequate function of cells against homeostatic disturbances such as those caused by septic shock, aging and, in general, processes involving oxidative stress. This review focuses on the involvement of reactive oxygen and nitrogen species.
The presence of free radicals in biological materials was discovered about 50 years ago. Today, there is a large body of evidence indicating that patients in hospital intensive care units (ICUs) are exposed to excessive free radicals from drugs and other substances that alter cellular reduction -oxidation (redox) balance, and disrupt normal biological functions. However, low levels of free radicals are also vital for many cell signaling events and are essential for proper cell function.
Excess free radicals can result from a variety of conditions such as tissue damage and hypoxia (limiting oxygen levels), overexposure to environmental factors (tobacco smoke, ultraviolet radiation, and pollutants), a lack of antioxidants, or destruction of free radical scavengers. When the production of damaging free radicals exceeds the capacity of the body’s antioxidant defenses to detoxify them, a condition known as oxidative stress occurs.

Free Radicals and Antioxidants: an Overview

A free radical can be described as any atom or a group of atoms or molecules in which there is at least one unpaired electron in the outermost shell . These free radicals are very reactive with adjacent molecules such as lipids, proteins, and carbohydrates and can cause cellular damage. Paradoxically, free radicals can also be produced by many cells as a protective mechanism, for example neutrophils produce free radicals to attack and destroy pathogens, while the liver uses free radicals for detoxification. However, the presence of free radicals within the body can also have a significant role in the development and progression of many disease processes for example heart disease, hypertension, cerebrovascular accidents, and diabetic complications. Any free radical involving O2 is referred to as a reactive oxygen species (ROS).
Normal cellular metabolism involves the production of ROS, and in humans, superoxide (O2 -) is the most commonly produced free radical. Phagocytic cells such as macrophages and neutrophils are prominent sources of O2 -. During an inflammatory response, these cells generate free radicals that attack invading pathogens such as bacteria and, because of this, the production of O2- by activated phagocytic cells in response to inflammation is one of the most studied free radical producing systems. The majority of the H2O2 is broken down to O2 and water by the antioxidant enzyme catalase. In addition to catalase, glutathione peroxidase can also break down H2O2 and also any peroxides that form on lipids within the body. When O2 – reacts with nitric oxide (NO), the toxic product peroxynitrite (ONOO-) is formed.
Cellular ROS originate from O2- generated as a by-product of oxidative phosphorylation (mitochondrial respiration), they differ in their mechanism of production, necessary cofactors, diffusion range, hydrophobicity, biological targets, detoxification pathways and breakdown products. O-2 damaging reactions largely involve disassembly of iron-sulphur clusters in proteins. H2O2 or O-2 alone lacked reactivity toward iron regulatory protein-1 (IRP-1), but a combined action of the two species induced reversible inactivation of IRP-1. Such an effect was attributed to direct interactions of O-2 and H2O2 with a preformed pool of IRP-1, resulting in reversible modifications of -SH residues; in fact, its action would be limited to removing only iron atoms, an effect sufficient to abolish enzyme activity.
The hydroxyl radical (.OH) is the most reactive of the free radical molecules. OH- damages cell membranes and lipoproteins by a process termed lipid peroxidation. In fact, lipid peroxidation can be defined as the process whereby free radicals “steal” electrons from the lipids in our cell membranes, resulting in cell damage and increased production of ROS. This process takes place in 3 stages:

1. Initiation: In a peroxide-free lipid system, the initiation of a peroxidation sequence refers to the attack of an ROS (with sufficient reactivity) able to abstract a hydrogen (H) atom from a methylene group (- CH2-).
2. Propagation: A peroxyl radical is able to abstract H from another lipid molecule (adjacent fatty acid), especially in the presence of metals such as copper or iron, thus causing an autocatalytic chain reaction. The peroxyl radical combines with H to give a lipid hydroperoxide (or peroxide).
3. Termination: formation of a hydroperoxide. Lipid peroxidative damage to lipids in low-density lipoprotein (LDL) plays an important role in atherosclerosis [9]. To protect against oxidative damage, organisms have developed a variety of antioxidant defenses that include proteins, compounds such as vitamins, and specialized antioxidant enzymes.

Lipid-soluble antioxidants are located in the cellular membranes and lipoproteins, whereas the water-soluble antioxidants are present in the aqueous environments, such as fluids inside cells and in the blood. Preventative antioxidant enzymes inside the cell are an important defense against free radicals.
In humans, the highest levels of SOD are found in the liver, adrenal gland, kidney, and spleen. Catalase and glutathione peroxidase both work to detoxify O2-reactive radicals by catalyzing the formation of H2O2 derived from O2 -. The liver, kidney, and red blood cells possess high levels of catalase, which helps to detoxify chemicals in the body. The water-soluble tripeptide-thiol glutathione also plays an important role in a variety of detoxification processes. Glutathione is found in millimolar concentrations in the cell cytosol and other aqueous phases, and readily interacts with free radicals, especially the hydroxyl radical, by donating a hydrogen atom.

Sepsis and Signaling Pathways

Serious infections trigger systemic inflammatory response and can result in sepsis. It is believed that sepsis and therefore septic shock are due to the inappropriate increase in the innate immune response via circulating and tissue inflammatory cells, such as monocytes/macrophages and neutrophils. These cells normally exist in a nonactivated state but are rapidly activated in response to bacteria. Sepsis induces a dysfunction in immune cells that contributes to the development of injuries by producing mediators such as cytokines and ROS.

Lipopolysaccharide (Lps) Signaling

LPS of Gram-negative organisms induces macrophages to secrete cytokines, which in turn activate T, and B cells to upregulate the adaptive immune responses. Toll-like receptor 4 (TLR4) is the LPS receptor and its stimulation induces nuclear factor kB (NF-kB) activation. The activation of NF-kB involves phosphorylation and degradation of IkB, an inhibitor of NF-kB. The NF-kB/IkB system exerts transcriptional regulation on proinflammatory genes encoded for various adhesion molecules and cytokines. Activation of NF-kB leads to the induction of NF-kB binding elements in their promoter regions and also leads to the induction of NF-kB dependent effector genes, which produce modifications in blood flow, and aggregation of neutrophils, and platelets. This results in damaged endothelium and also coagulation abnormalities often seen in patients with sepsis and septic shock. Therefore, NF-kB is reported to be an O2 sensor in LPS-induced endotoxemia.

Free Radicals and Antioxidants In Sepsis

The sources of ROS during sepsis are:

1. the mitochondrial respiratory chain.
2. the metabolic cascade of arachidonic acid.
3. the protease-mediated enzyme xanthine oxidase.
4. granulocytes and other phagocytes activated by complement, bacteria, endotoxin, lysosomal enzymes, etc.
5. Other oxidases mainly NADPH oxidase.

Under normal physiological conditions, the majority of ROS are formed during cellular respiration and by activated phagocytic cells, including neutrophils, involved in the inflammatory response. ROS have physiologically essential roles in mitochondrial respiration, prostaglandin production pathways and host defense . The electron reduction of O2 occurs in the mitochondrial electron transport system of all aerobically respiring cells. The enzyme catalyzing this transition metals iron and copper in its active site. These ions can be paramagnetic and contain stable unpaired electrons. By using the unpaired electrons in these transition metals to control the O2 reactions, mitochondria prevent the unwanted release of ROS.
In sepsis, there are several potential sources of ROS, including the mitochondrial respiratory electron transport chain, xanthine oxidase activation as a result of ischemia and reperfusion, the respiratory burst associated with immune cell activation, arachidonic acid metabolism and NADPH oxidase.

• In fact, activated immune cells produce O2 – as a cytotoxic agent as part of the respiratory burst via the action of membrane-bound NADPH oxidase on O2.
• The increase of ROS after LPS challenge has been demonstrated in different models of septic shock in peritoneal macrophages and lymphocytes.

This disturbance in the balance between pro-oxidants (ROS) and antioxidants in favor of the former is characteristic of oxidative stress in immune cells in response to endotoxin. In this context,

• a typical behavior of these cells under an oxidative stress situation implies changes in different immune functions such as an increase in adherence and phagocytosis and a decrease in chemotaxis.
• Neutrophils play a crucial role in the primary immune defense against infectious agents,which includes phagocytosis and the production of ROS.

Antioxidant Defenses

Antioxidants are central to the redox balance in the human body. They do not act in isolation, but synergistically with other classes of molecules. Primary antioxidants prevent oxygen radical formation, by either removing free radical precursors or by inhibiting catalysis, e.g. the enzymes glutathione peroxidase and catalase. Secondary antioxidants react with ROS which have already been formed, either to remove or inhibit them, e.g. vitamins C and E. Endogenous antioxidant defenses exist in a number of locations, namely intracellularly, on the cell membrane and extracellularly. The immune system is highly reliant on accurate cell-cell communication for optimal function, and any damage to the signaling systems involved will result in an impaired immune responsiveness.

• Oxidant-mediated tissue injury is a particular hazard to the immune system, since phagocyte cells produce ROS as part of the defense against infection.
• Therefore, adequate amounts of neutralizing antioxidants are required to prevent damage to the immune cells themselves.

The SOD enzymes are a family of metalloenzymes which rapidly promote the conversion of O2- to H2O2. Three forms of SOD are recognized to be important: copper-zinc SOD (cytoplasmic-located), manganese SOD (mitochondrial-located) and extracellular SOD (extracellular matrix-located). Catalase and glutathione peroxidase, a selenium containig enzyme which requires the presence of reduced GSH for its action, both catalyze the conversion of H2O2 to H 2O. GSH also has direct antioxidant activity, through donation of hydrogen ions, to repair damaged DNA. Oxidative stress and modulation on GSH/GSSG (GSSG=oxidized GSH) levels also up-regulate gene expression of several other antioxidant proteins, such as manganese SOD, glutathione peroxidase, thioredoxin (Trx) and metallothionein.

Effects of Nitric Oxide

NO is synthesized from L-arginine by different isoenzymes of (NOS), and is implicated in a wide range of disease processes, exerting both detrimental and beneficial effects at the cellular and vascular levels. To date, three main isoforms of NOS are known:

• neuronal NOS (NOS-1 or nNOS),
• inducible NOS (NOS-2 or iNOS), and
• endothelial NOS (NOS-3 or eNOS).

NO has been shown to play a key role in the pathogenesis of septic shock

Hyperproduction of NO induces

• excessive vasodilation,
• changes in vascular permeability, and
• inhibition of noradrenergic nerve transmission,

all characteristics of human septic shock.
The recogniton of NO production by activated macrophages as part of the inflammatory process was an important milestone for assesing both the biological production of NO and the phenomenon of induction of NOS activity. The observation has been extended to neutrophils, lymphocytes, and other cell types. The role of NO in the pathophysiology of endotoxic shock was advanced by Thiemermann and Vane, who observed that administration of the specific NOS inhibitor N-methyl-L-arginine (L-NMMA) decreased the severe hypotension produced by administration of LPS. Other groups simultaneously reported similar results indicating that endotoxin increases NO production and prompted the idea that pharmacological inhibition of NOS may be useful in the treatment of inflammation and septic shock. However, clinical trials using L-NMMA failed to show a beneficial effect in septic shock patient. The major limitation for the use of NOS inhibitors in clinical studies is the development of pulmonary hypertension as a side effect of NOS blockade, which can be alleviated by the use of inhaled NO.
However, several compounds which modulate NO synthesis have been patented in recent years, such as various inflammatory mediators that have been implicated in the induction and activation of iNOS, particularly IFNg, TNFa, IL-1b, and platelet-activating factor (PAF) alone or synergistically. In addition to the activation of iNOS, cytokines and endotoxin may increase NO release by increasing arginine availability through the opening of the specific y+ channels and the expression of the cationic amino acid transporter (CAT), or by increasing tetrahydrobiopterin levels, a key cofactor in NO synthesis. Several experimental studies have demonstrated a decrease in NOS activity resulting in an impairment in endothelial-dependent relaxation during endotoxemia and experimental sepsis, possibly as the result of a cytokine-or hypoxia-induced shortened half-life of NOS mRNA, or of altered calcium mobilization.
NO exerts in vitro toxic effects including nuclear damage, protein and membrane phospholipid alterations, and the inhibition of mitochondrial respiration in several cell types. Mitochondrial impairment could also be considered as an adaptive phenomenon, decreasing cellular metabolism when the energy supply is limited. The toxicity of NO itself may be enhanced by the formation of ONOO- from the reaction of NO with O-2. Therefore, the multiple organ failure syndrome (MOFS) that often accompanies severe sepsis may be related to the cellular effects of excess NO or ONOO-.

Involvement of Nitrogen Species

NO reacts rapidly with ferrous iron, and at physiological concentrations, NO also binds to soluble guanylate cyclase and to another hemoprotein, cytochrome c oxidase (Complex IV), the terminal enzyme of the mitochondrial respiratory chain. NO can therefore control cellular functions via the reversible inhibition of respiration. There are a number of reactive NO species, such as

• N2O3 and
• ONOO-

that can also alter critical cellular components.

During the first hours after injury, iNOS-mediated NO production is upregulated, producing a burst of NO that far exceeds basal levels. This overabundance of NO produces significant cellular injury via several mechanisms.
NO may

• directly promote overwhelming peripheral vasodilation, resulting in vascular decomposition;
•  NO may upregulate the transcription NF-kB initiating an inflammatory signaling pathway that, in turn,
• triggers numerous inflammatory cytokines.

NO also interacts with the O-2 to yield ONOO-, a highly reactive compound that exacerbates the injury produced by either O-2 alone or NO alone.
The ONOO- generation which occurs during fluid resuscitation in the injured subject produces cellular death by enhancing DNA single strand breakage, activates the nuclear enzyme polyADP ribose synthetase (PARS), leading to cellular energy depletion and cellular necrosis. The detrimental effects of ONOO- in shock and resuscitation have been attributed to oxidation of sulfhydryl groups, the nitration of tyrosine, tryptophane, and guanine, as well as inhibition of the membrane sodium-potassium adenosine triphosphatase. PARS activation depletes NAD and thus alters electron transport, ATP synthesis, and glycolysis; and leads to DNA fragmentation and cellular apoptosis.
The activation of monocytes, macrophages and endothelial cells by LPS results in the expression of iNOS, and consequently increases the transformation of L-arginine to NO, which can combine with O2- to form ONOO-, causing tissue injury during shock, inflammation and ischemia reperfusion. NO stimulates H2O2 and O-2 production by mitochondria, increasing leakage of electrons from the respiratory chain. H2O2, in turn, participates in the upregulation of iNOS expression via NFkB activation. ONOO- has been shown to stimulate H2O2 production by isolated mitochondria. On the other hand, NO can decrease ROS-produced damage that occurs at physiological levels of NO. The high reactivity of NO with radicals might be beneficial in vivo by scavenging peroxyl radicals and inhibiting peroxidation. ONOO- may also be a signal transmitter and can mediate vasorelaxation, similarly to NO.
Local generation of RNS contributes to tissue injury. Recent studies have demonstrated that activation of the nuclear enzyme poly(ADP-ribose) polymerase-1 by RNS-mediated DNA damage is an important pathway of tissue injury in conditions associated with oxidative stress. Increased formation of RNS in response to endotoxin challenge is organ specific.
In sepsis, NO may exert direct and indirect effects on cardiac function. Sustained generation of NO occurs in systemic inflammatory reactions, such as septic shock with involvement in circulatory failure. In fact, myocardial iNOS activity has been reported in response to endotoxin and cytokines and inversely correlated with myocardial performance. Low-to-moderate doses of iNOS inhibitors restore myocardial contractility in hearts exposed to proinflammatory cytokines, whereas at higher doses, the effects are reversed. This finding may indicate that small amounts of NO produced by iNOS may be necessary to maintain contractility and can be cardio-protective in experimental sepsis.

Nitric oxide in Septic Shock

A list of effects of NO in sepsis is as follows.

• Inhibition of nitric oxide synthesis causes myocardial ischemia in endotoxemic rats
• Nitric oxide causes dysfunction of coronary autoregulation in endotoxemic rats
• Prolonged inhibition of nitric oxide synthesis in severe septic shock
• Effect of L-NAME, an inhibitor of nitric oxide synthesis, on cardiopulmonary function in human septic shock
• Pulmonary hypertension and reduced cardiac output during inhibition of nitric oxide synthesis in human septic shock
• Effect of L-NAME, an inhibitor of nitric oxide synthesis, on plasma levels of IL-6, IL-8, TNF-u and nitrite/nitrate in human septic shock
• Endothelin-1 and blood pressure after inhibition of nitric oxide synthesis in human septic shock
• Distribution and metabolism of NO-nitro-L-arginine methyl ester in patients with septic shock

The possible involvement of the L-arginine-NO pathway in both the vascular and cellular processes seen in sepsis has been supported by numerous in vitro and in vivo studies. iNOS appears to be expressed in a wide array of cell types during sepsis, including immune cells (such as macrophages, neutrophils, T lymphocytes), as well as cells outside the classical immune system (for example, hepatocytes, Kuppfer cells, vascular smooth muscle cells, endothelial cells, and fibroblasts). Expression of iNOS is regulated, both positively and negatively, by a number of mediators present during infection and inflammation. The main stimuli for iNOS induction indude lipopolysaccharide (LPS), interferon-y, interleukin (IL)-10, and tumor necrosis factor (TNF)-a; inhibitory cytokines, such as transforming growth factor-5, IL-4 and IL-10, as well as glucocorticoids, can prevent this induction. The expression of iNOS in response to these agents differs among cell types, but a maximal inducing effect is generally obtained by the combination of microbial products and cytokines acting synergistically.

iNOS activity is also regulated by substrate and cofactor availability. Tetrahydrobiopterin (BH4), an essential cofactor for the enzyme, is coinduced with iNOS in cytokine-stimulated vascular smooth muscle cells.
NO is a simple molecule, but its widespread production in sepsis, coupled with its effects on a variety of intracellular and extracellular target molecules, results in a complex array of biologic roles. Interaction of NO with the metalloproteins in a number of key enzymes can modulate their activity. Many of the signaling actions of ‘NO are mediated by soluble guanylate cyclase. By binding the iron on the heme component of soluble guanylate cyclase, NO is able to activate the enzyme leading to cyclic guanosine monophosphate (cGMP) formation.

Increased cGMP levels account for several of the important cellular actions of NO, including

• smooth muscle relaxation,
• platelet aggregation and adherence, as well as
• neutrophil chemotaxis.

However, *NO can adversely affect cellular metabolism through its disruption of iron-sulfur clusters in essential energy-generating enzymes involved in mitochondrial electron transport, glycolysis, and the Krebs cycle. Further, high concentrations of induced macrophage produced NO can directly interfere with DNA in target cells, resulting in fragmentation.
Another critical reaction that ‘NO undergoes during inflammation is with the superoxide anion radical (02j, yielding peroxynitrite (OONO-). OONO- is a potent oxidant that can decay under acidic conditions to produce a powerful hydoxyl-like free radical. This reaction between *NO and O2 can have a protective or damaging consequence, depending on the individual sites and rates of production of the free radicals, and the redox status of both the generating cells as well as the target cells. OONO- formation can initiate adverse effects such as lipid peroxidation of membranes, and modification of structural proteins through nitration of tyrosine residues (14). Indeed, increased levels of 3-nitrotyrosine have been detected in the lungs of patients with sepsis and animals with acute lung injury. However, OONO- can also S-nitrosylate glutathione and other thiol-containing substances to form S-nitrosothiols, which have marked cardioprotective and cytoprotective effects.
The damaging effect of NOS inhibition may be, in part, mediated by oxygen radicals and platelet deposition, suggesting a cytoprotective role of NO in preventing microvascular thrombosis and as a free radical scavenger. In addition, ‘NO has a protective role in hepatic microcirculatory dysfunction during sepsis through its effect on leukocyte adherence to sinusoidal walls. ‘NO may also protect against circulatory vasoconstrictors during inflammation, as enhanced ‘NO synthesis counteracted phenylephrine-induced increases in intrahepatic resistance in endotoxin-treated rats. Finally, we have recently demonstrated that different types of NOS inhibitors resulted in detectable apoptosis in the liver following LPS injection. This increase in apoptosis was present even with L-N-iminoethyl-lysine (L-NIL), a rather specific inhibitor of iNOS, revealing another important protective role of NO as an antiapoptotic agent in sepsis.
Even though overproduction of *NO in the vasculature contributes to the vasodilatation seen in septic shock, iNOS expression during inflammation also represents a beneficial, adaptive response in some organ systems. Moreover, different tissues can react dissimilarly to the effects of ‘NO cytotoxicity. In this setting, global nonselective inhibition of NOS, including the potentially undesirable consequences of eNOS inhibition, would be harmful. If confirmed, this would suggest that use of isoform-specific inhibitors of NOS within the vascular bed would be more appropriate.

Pulmonary Hypertension and Reduced Cardiac Output

Pulmonary hypertension and reduced cardiac output can be major side effects of continuous NO synthase inhibition. Pulmonary vasoconstriction is undesirable because it may compromise pulmonary gas exchange and because it increases the workload on the right ventricle. In cases where strain already exists on the right ventricle (e.g. sepsis or PEEP ventilation) or in cases where right sided cardiac reserve is minimal, such increase in workload may lead to right ventricular failure, reduced cardiac output and compromised tissue perfusion.
Blood pressure and systemic vascular resistance increased during infusion of the NO synthase inhibitor L-NAME, and the dosage of catecholamines was reduced. The vasoconstrictive response to L-NAME most likely was the result of blocking the NO system . In addition to the systemic effects of L-NAME, severe pulmonary vasoconstriction was observed with L-NAME. Analogous to these findings, in patients with Adult Respiratory Distress Syndrome (ARDS), inhalation of NO is reported to be beneficial by causing local vasodilation in bronchial and pulmonary circulation which results in reduced pulmonary vascular resistance and improved oxygenation. This suggests that the pulmonary circulation is sensitive to the vasodilating effects of both endogenous and exogenous NO. Pulmonary vasoconstriction is not, therefore, unexpected with systemic inhibition of NO synthesis. With a continuous infusion of L-NAME, pulmonary vascular resistance increased five-fold, whereas systemic vascular resistance “only” doubled. pulmonary hypertension was reversible after stopping L-NAME infusion. In prior experiments with a lower dose of LNAME, pulmonary vasoconstriction was less pronounced and did not result in pulmonary hypertension.’ Thus, pulmonary hypertension is a dose-related effect of L-NAME that can probably be attributed to overdosing of the drug. Reduced cardiac output may have directly resulted from the extreme increase in pulmonary vascular resistance compromising venous return and left ventricular preload and/or a reflex reduction in heart rate by the increase in vascular resistance and blood pressure.
S-methyl-isothiourea, a relatively selective inhibitor of iNOS activity, decreased pulmonary leak and improved survival in endotoxemia. However, because of the tissue-protective and antiapoptotic effects of NO, even selective iNOS inhibitors may be detrimental in certain tissues during sepsis.Combining the salutary effects of site-specific local donors that exploit the cytoprotective actions of ‘NO with specific agents that combat the deleterious hypotensive and tissue-damaging effects of ‘NO overproduction may be needed to treat septic shock. In this regard, inhaled ‘NO gas has shown promise as a selective pulmonary vasodilator.

Role of Nitric Oxide in Inflammation and Tissue Injury

Since the discovery that nitric oxide (‘NO) accounts for the biologic activity of endothelial-derived relaxing factor, a torrent of research over the last decade has focused on its role, protective or detrimental, in myriad pathophysiologic conditions. Recently, increasing attention has focused on ‘NO as a possible mediator of the severe hypotension and impaired vasoreactivity characteristic of circulatory failure. Experimental and clinical studies have suggested NOS inhibition might have therapeutic potential in circulatory shock, and other studies have demonstrated the beneficial nature of iNOS expression in modulating tissue perfusion and mediating cytotoxicity. However, inhibition of ‘NO synthesis in experimental and clinical studies of shock has yielded mixed, sometimes contradictory, results. Overproduction of ‘NO in the vasculature may result in systemic vasodilatation, but still ‘NO synthesis has a beneficial role in regulating organ perfusion and mediating cytotoxicity.

Diminished *NO Production Occurs with Hemorrhage

These findings are consistent with those in trauma patients, where nitrite and nitrate levels were reduced for prolonged periods after injury. This impairment of ‘NO production in victims of hemorrhagic hypotension may be due to impairment of eNOS, and indeed, several investigators have demonstrated decreased vasodilatory activity in vascular rings taken from hemorrhaged animals in response to agonists that stimulate endothelial ‘NO production. In studies of hemorrhagic shock no iNOS expression could be detected until the very late irreversible phase of HS. The hemodynamic instability associated with decompensation occurred well before NOS induction.
Using either the selective inhibitor L-NIL or iNOS knockout mice, iNOS inhibition or deficiency not only prevented the upregulation of the inflammatory cytokines IL-6 and granulocyte colony-stimulating factor following resuscitation from HS but also produced a marked reduction in lung and liver injury. Furthermore, the activation of the proinflammatory transcriptional factors nuclear factor kappa B and signal transducer and activator of transcription 3 was also reduced, suggesting iNOS upregulation has a key role in proinflammatory signaling and the subsequent activation of inflammatory cascades. Recent studies have implicated a possible redox-sensitive mechanism. ‘NO activates the critical signaling enzyme p21ras through S-nitrosylation.
Vascular quenching of ‘NO using scavengers may again provide an alternative to NOS inhibition as a means to achieve the goal of reducing ‘NO levels. Use of ‘NO scavengers after HS and resuscitation may serve to supplement a possibly depleted antioxidant defense system and limit the harmful effects of free radicals such as OONO- and hydoxyl radicals. Removal of ‘NO by this method is complicated by the extreme rapidity of the reaction between ‘NO and 02’- .
iNOS upregulation also has a beneficial protective role in several organ systems. In conditions where excess NO production results in maladaptive damaging consequences with disruption of homeostasis, the therapeutic strategy should be to remove this surplus ‘NO without adversely affecting the cytoprotective actions of *NO. Interfering with the physiologic and microcirculatory role of eNOS through nonselective, global inhibition of NOS is undesirable in shock.

Effects of nitric oxide in endotoxemia and hemorrhagic shock and proposed therapeutic strategies for manipulation of nitric oxide production.

Endotoxemia Hemorrhagic shock
Effects of NO                         Beneficial                            Beneficial
by eNOS                      -maintains perfusion;    -maintains perfusion;
cytoprotective               cytoprotective
iNOS                             Beneficial and toxic-         Beneficial and toxic-

depending on site can induce tissue damage and  of production and promote inflammation with microenvironment sustained shock

Therapeutic strategy

Inhibition of eNOS                Avoid                                     Avoid
Inhibition of iNOS         Possibly desirable-       Probably desirable-
to reduce                          to limit exaggerated inflammatory
cytotoxicity and           response and development of
combat hypotension       MODS

NO scavengers   Probably desirable-quench    Probably desirable-quench
extracellular NO without        extracellular NO without
inhibition of eNOS or iNOS;    inhibition of eNOS or iNOS;
supplement antioxidant defenses       supplement antioxidant defenses
NO donors          Possibly desirable-site-            Possibly desirable-site-
specific donors without adverse     specific donors without adverse
systemic  side effects;                      systemic side effects;
limited availability                             limited availability

Therapeutic Outlook

LINCS: L-NAME (a NO synthase inhibitor)
Patients were randomized to supportive care alone (n=15, control group) or to supportive care in addition to L-NAME (1 mg/Kg bolus and 1 mg/Kg/h continuous IV drip for 5 h n=15). Death at one month was 27% in the L-NAME group vs. 67% in the control group (p=0.008). Time on IABP and time on mechanical ventilation were significantly shorter in the L-NAME group. The results of this study indicate that NO synthase inhibitors are beneficial in the treatment of patients with refractory cardiogenic shock.
Inducible Nitric Oxide Synthase Inhibitors
Inducible nitric oxide synthase (iNOS)-dependent production of nitric oxide (NO) plays an important role in inflammation. The effects of various naturally occurring furanocoumarins on NO production in lipopolysaccharide (LPS)-activated RAW 264.7 macrophage cells were evaluated in vitro. The results showed that angelicin, pimpinellin, sphondin, byakangelicol, oxypeucedanin, oxypeucedanin hydrate, xanthotoxin, and cnidilin are potential NO production inhibitors, and their IC50 values for inhibition of nitrite production were 19.5, 15.6, 9.8, 16.9, 16.8, 15.8, 16.6, and 17.7 mg/mL, respectively.

Distinct structure activity relationships were also revealed for the NO production inhibitory activities of these furanocoumarins. Activities of the angelicin type such as pimpinellin and sphondin were more potent than those of the psoralen type. Presence of a methoxy at the C6 position in the angelicin type seemed to be essential to augment the activity. Western blot analysis demonstrated that only sphondin dose-dependently inhibited the expression of the iNOS protein at 2.5±20 mg/mL. However, iNOS enzyme activity was stimulated with LPS for 12 h and sphondin was administered (20 mg/mL) for 24 h, which did not reasonably inhibit iNOS enzyme activity. l-NAME (100 mM), a known specific inhibitor of iNOS, was employed as a positive control with the same protocol and showed more than 50% inhibition activity. The results demonstrate that the NO production inhibitory activity of sphondin is due to the effect of iNOS expression, but not by direct inhibition of iNOS enzyme activity. Thus, sphondin may act as a potent inhibitor of NO production under tissue-damaging inflammatory conditions.
S-Methylisothiourea Sulfate, A Potent And Selective Inhibitor Of Inducible Nitric Oxide Synthase
Non-isoform-selective inhibition of NO formation, however, may lead to side effects by inhibiting the constitutive isoform of NOS and, thus, the various physiological actions of NO. S-Methylisothiourea sulfate (SMT) is at least 10- to 30-fold more potent as an inhibitor of inducible NOS (iNOS) in immuno-stimulated cultured macrophages (EC50, 6 ,AM) and vascular smooth muscle cells (EC50, 2 ,uM) than NG-methyl-L-arginine (MeArg) or any other NOS inhibitor yet known. The effect of SMT on iNOS activity can be reversed by excess L-arginine in a concentration-dependent manner.

Enhanced formation of NO following the induction of iNOS contributes importantly to the circulatory failure (hypotension and vascular hyporeactivity to vasoconstrictor agents) in circulatory shock of various etiologies.
SMT dose-dependently reverses (0.01-3 mg/kg) the hypotension and the vascular hyporeactivity to vasoconstrictor agents caused by endotoxin [bacterial lipopolysaccharide]
SMT, a potent and selective inhibitor of iNOS, may have considerable value in the therapy of circulatory shock of various etiologies and other pathophysiological conditions associated with induction of iNOS.
SMT, or other iNOS-selective inhibitors, are likely to have fewer side effects which are related to the inhibition of eNOS, such as excessive vasoconstriction and organ ischemia), increased platelet and neutrophil adhesion and accumulation, and microvascular leakage.

Iron Chelates Bind Nitric Oxide

Nitric oxide (NO), a short-lived potent vasodilator, was first described as the endothelium-derived relaxation factor (EDRF). The formation of NO from the guanidine nitrogen group of L-arginine is catalyzed by a group of enzymes termed constitutive (cNOs) and inducible (iNOs) NO synthases. The inducible form is not present constitutively in mammalian cells but is induced by proinflammatory stimuli such as bacterial lipopolysaccharide (LPS), Corynebacterium parvum, and the cytokines tumor necrosis factor-a, interleukin-1, or interferon-y, individually or in combination. Excess production of NO is reported to be associated with the development of hypotension associated with endotoxemia and sepsis.

Electrochemical studies show that FeIII-(DTPA)2- binds NO stoichiometrically upon reduction to iron(II) at biologically relevant potentials to form a stable NO adduct. In contrast, FeI”I(HDFB)+ is a stable and efficient electrocatalyst for the reduction of NO to N20 at biologically relevant potentials. These results suggest that the mechanism of protection against death by septic shock involves NO scavenging and that particularly effective drugs that operate a low dosages may be designed based on the principle of redox catalysis. These complexes constitute a new family of drugs that rely on the special ability of transition metals to activate small molecules.

Iron complexes could act as general NO scavengers and provide protection against septic shock. Iron complexes are capable of forming relatively stable NO adducts. Metal complexes, and in particular iron chelators, could act as “molecular sponges,” mopping up the excess NO produced during septic shock. Iron chelators can sequester and (as for 2) catalyze conversion of NO to benign products. Demonstration of mechanistic aspects of septic shock protection in vivo, including interaction with other free radicals, may be hampered by the detection limits of current analytical techniques. To detect the NO Fe-DETC complex formation in livers of LPS-treated mice by the electron paramagnetic resonance.

After screening a library of metal chelators and chelates [Fe(III)(H2DTPA)] and [Fe(III)(HDFB)]+ offered the highest mortality decrease in an experimental model of septic shock. The Fe(II) form of both complexes can bind NO, which appears to be related to their biological function.
Survival was greatly enhanced by the administration of 4 or 2 either 2 h before and at the time of or 30 min after LPS. In contrast, the Fe3+-free ligands of these compounds, 3 and 1, were less protective when administered before and at the time of LPS and virtually ineffective when administered after LPS. The clear advantage of 4 over 2 when administered after LPS was observed over a large number of experiments [76% survival with 4 (n = 102 mice) and 38% survival with 2 (n =64 mice)].

The hydroxamic acid siderophore ferrioxamine B [Fe”‘(HDFB)+] and the iron complex of diethylene-triamine-pentaacetic acid [FeI”(DTPA)2i] protected mice against death by septic shock induced by Corynebacterium parvum + lipopolysaccharide. Although Fem(DTPA)2- was somewhat more effective than FeI”(HDFB)+, the iron-free ligand H4DFB+ was significantly more effective than DTPA. The hydroxamic acid chelator has a much higher iron affinity than the amine carboxylate, allowing for more efficient formation of the FeI”(HDFB)+ complex upon administration of the iron-free ligand.

Efficacy of Treatment With the Iron (III) Complex of Diethylenetriamine Pentaacetic Acid

Bacteremia and septic shock also are associated with overproduction of free radicals such as hydroxyl, superoxide, and carbon- and oxygen-centered radicals. In addition, nitric oxide (NO) overproduction is at least partly responsible for the vasodilation that causes a reduction in mean systemic arterial pressure (MSAP) and organ perfusion pressure during septic shock. This overproduction of NO likely results from early activation of the endothelial constitutive form of NO synthase followed by induction of the inducible form of NO synthase via TNF and IL-1.
The simultaneous increase and further reaction of NO with superoxide, which yields the oxidant peroxynitrite anion, occurs in cellular systems in response to inflammatory mediators. In addition, in sepsis-associated adult respiratory distress syndrome (ARDS), the presence of nitrotyrosine residues (formed by reaction of peroxynitrite and the tyrosine residues of proteins) are apparent throughout the lung.

Administration of the iron (III) complex of diethylenetriamine pentaacetic acid (DTPA iron (III), prevented death in Corynebacterium parvum 1 LPS-treated mice. Using electrochemistry, the binding of NO to DTPA iron (II) is confirmed. The DTPA iron (II) form can be easily formed by common biological reductants, because the potential for the iron (III/II) couple is E = 0.22.
Treatment with DTPA iron (III) resulted in a significant decrease in mortality compared to the untreated controls. The efficacy of DTPA iron (III) increased when given to mice 2 h or more after infection. The best results were observed when DTPA iron (III) was given 5 h after infection.

The iron (III) complex of diethylenetriamine pentaacetic acid (DTPA iron [III]) protected mice and baboons from the lethal effects of an infusion with live LD 100 Escherichia coli. In mice, optimal results were obtained when DTPA iron (III) was administered two or more hours after infection. Prevention of death occurred in spite of the fact that the adverse effects of TNFa were well underway in the mouse model.
In septic baboons, survival was observed after administration of two doses of DTPA iron (III) at 2.125 mg/kg, the first one given before, or as late as 2 h after, severe hypotension. Administration of DTPA iron (III) did not alter mean systemic arterial pressure, but did protect baboons in the presence of high levels of TNFa and free radical overproduction. Furthermore, exaggerated production of nitric oxide was attenuated. Because of its ability to interact in vitro with free radicals, its poor cell permeability, and its short half-life, we postulate that DTPA iron (III) and/or its reduced form may have protected the mice and baboons by sequestration and subsequent elimination of free radicals (including nitric oxide) from their systems. (J. Clin. Invest.1996. 98:192–198.)
Inhibitor of Poly(Adenosine 5′-Diphosphate-Ribose) Synthetase
Poly(adenosine 5′-diphosphate [ADP]-ribose) synthetase (PARS) is a nuclear enzyme which, when activated by DNA singlestrand breaks, initiates an energy-consuming, inefficient metabolic cycle by transferring ADP-ribose units to nuclear proteins. The result of this process is a rapid depletion of intracellular oxidized nicotinamide adenine dinucleotide and adenosine 5′-triphosphate energetic pools, which slows the rate of glycolysis and mitochondrial respiration, leading to cellular dysfunction and death.
Reactive oxygen-centered radicals (superoxide, hydroxyl radicals, singlet oxygen, and hydrogen peroxide) and peroxynitrite (a reactive oxidant produced from the reaction of superoxide and nitric oxide) are powerful triggers of DNA single strand breakage, and they induce activation of a cell suicide cycle governed by PARS in various cell types in vitro.

Multiple reports implicated a role of PARS activation in the pathophysiology of endotoxic shock, hemorrhagic shock, and various forms of ischemia-reperfusion injury.
Twenty pigs were chronically instrumented with intracardiac transducers to measure left ventricular pressure, sonomicrometer crystals in the left ventricle to measure short axis diameter, an ultrasonic flow meter to measure cardiac output, and catheters in the pulmonary artery and aorta to measure blood pressures and collect samples. By using a randomized study design, either the novel potent PARS inhibitor PJ34 (10 mg/kg for 1 hr, 2 mg·kg 1·hr 1 for 96 hrs) or placebo to pigs immediately before intraperitoneal implantation of Escherichia coli 0111.B4 (2.3 0.1 1010 colony-forming units/kg)-laden fibrin clots to produce peritonitis and bacteremia.
PJ34 treatment significantly attenuated this cytokine response. The formation of peroxynitrite and the activation of PARS were confirmed in hearts and lungs of the septic pigs by the immunohistochemical detection of nitrotyrosine and poly(ADP-ribose), respectively. Inhibition of PARS with PJ34 abolished poly(ADP-ribose) formation in septic animals.

Cardiac inotropicity was evaluated by analysis of percentage of short axis diameter shortening (one-dimensional ejection fraction). Bacteremia induced a rapid and progressive loss of inotropy until death in vehicle-treated pigs. A similar decline was observed in the first 6 hrs in PJ34 pigs. This decline was reversed on all subsequent days. Control pigs exhibited rapid and significant increases in systemic vascular (SVR) and pulmonary vascular (PVR) resistances.
This experimental model mimics many aspects of the human sepsis syndrome. Therefore, the positive survival benefit of PARS inhibition suggests a potential utility of PARS inhibitors in human sepsis management. PARS activation is triggered by DNA single-strand breakage.

The current work, demonstrating increased poly(ADP-ribose) staining in the heart of septic pigs, may point toward the importance of a myocardial, PARS dependent cardiodepressive mechanism in the current model of shock. This hypothesis is supported by the following findings:

• free radicals cause myocardial dysfunction and injury in a PARS dependent fashion in vitro;
• in the current study, pharmacologic inhibition of PARS with PJ34 markedly improved myocardial function; and

in prior studies, pharmacologic inhibition of PARS markedly improved myocardial contractile function in hypoxic-reoxygenated hearts as well as in a porcine model of hemorrhagic shock.
Treatment with a potent PARS inhibitor improved survival and cardiovascular status and attenuated an important mediator component of the inflammatory response in a lethal porcine model of sepsis. (Crit Care Med 2002; 30:974 –980).

Decrease of the inflammatory response and induction of the Akt/protein kinase B pathway by poly-(ADP-ribose) polymerase 1 inhibitor

The lack of efficacy of anti-inflammatory drugs, anti-coagulants, anti-oxidants, etc. in critically ill patients has shifted interest towards developing alternative treatments. Since inhibitors of the nuclear enzyme poly-(ADP-ribose) polymerase (PARP) were found to be beneficial in many pathophysiological conditions associated with oxidative stress and PARP-1 knock-out mice proved to be resistant to bacterial lipopolysaccharide (LPS)-induced septic shock, PARP. The mechanism of the protective effect of a potent PARP-1 inhibitor, PJ34 was studied in LPS-induced (20 mg/kg, i.p.) septic shock in mice.

We demonstrated a significant inflammatory response by magnetic resonance imaging in the dorsal subcutaneous region, in the abdominal regions around the kidneys and in the inter-intestinal cavities. We have found necrotic and apoptotic histological changes as well as obstructed blood vessels in the liver and small intestine. Additionally, we have detected elevated tumor necrosis factor-a levels in the serum and nuclear factor kappa B activation in liver of LPS-treated mice.

Pre-treating the animals with PJ34 (10 mg/kg, i.p.), before the LPS challenge, besides rescuing the animals from LPS-induced death, attenuated all these changes presumably by activating the phosphatidylinositol 3-kinase–Akt/protein kinase B cytoprotective pathway.

PJ34, a novel, potent PARP-1 inhibitor was found to protect against LPS induced tissue damage. PARP inhibitors protected Langendorff-perfused hearts against ischemia-reperfusion induced damages by activating the PI3-kinase–Akt pathway. The importance of the PI3-kinase–Akt pathway in LPS induced inflammatory mechanisms has gained support, raising the question whether this pathway was involved in the effect of PJ34 on LPS-induced septic shock.
Among all the observed LPS-induced inflammatory responses, we found the most characteristic and most pronounced increases in the gastro-intestinal tract, but no signal increase could be observed inside the kidneys and in skeletal muscle, in the paravertebral or in the femoral muscle. All increases in signal intensities were significantly attenuated in mice treated with PJ34.

Effect of PJ34 on survival of LPS-treated mice

• PARP-1 inhibitor significantly protected the animals against LPS-induced death, with 86 and 43% surviving mice, respectively.
• PJ34 treatment itself did not induce death or any obvious damage.

Effect of PJ34 on LPS-induced NF-kB activation

LPS treatment in the lung caused a significant increase in NF-kB activation that was slightly but not statistically significantly attenuated by PJ34 pre-treatment.
in contrast to the lung, NF-kB activation in the liver was prevented by PJ34 pre-treatment
The other tissue with observable LPS-induced pathological changes was the small intestine. Atrophy of villi may reflect the diarrhea observed in the LPS-treated animals and is in agreement with the results of Abreu et al. who found a Fas-mediated apoptosis in intestinal epithelial cells that was sensitised by inhibitors of PI3-kinase and opposed by expressing constitutively active Akt.
Pre-treatment of the animals with a novel, potent PARP-1 inhibitor, PJ34, diminished the thoracic and abdominal inflammatory responses as revealed by T2 imaging, and abolished the above mentioned pathological changes.
The protective role of PARP inhibitors in septic shock is likely to be more complex than merely the regulation of NF-kB/Rel-dependent gene expression.
Activation of the PI3-kinase–Akt/protein kinase B cytoprotective pathway is likely to contribute to the protective effects of PARP inhibitors in shock and inflammation.

Carboxy-PTIO On Hemodynamic And Blood Gas Changes

Infusion of LPS caused a marked decrease in mean arterial pressure (MAP), metabolic acidosis, and hypoxia. These effects were reversed by co-administration of carboxy-PTIO, without affecting other hemodynamic parameters. In control animals, neither hemodynamic nor blood gas parameters changed with or without carboxy-PTIO.
These results indicate that carboxy-PTIO attenuates

• LPS-induced hypotension,
• metabolic acidosis, and
• hypoxia

by scavenging excess NO from the circulation without affecting NO synthase (NOS) activity. An NO scavenger, carboxy-PTIO, may be preferable to non-selective NOS inhibitors for the treatment of human septic shock.

Asymmetrical Dimethyl Arginine Levels

Overwhelming infection with resultant multiple organ failure, which has been termed the ‘sepsis syndrome’ , is a devastating illness with an incidence of 3 per 1,000 population per annum. It has been characterised as a dysregulation of inflammation in response to infection attributable to a combination of

• excessive inflammation,
• disseminated coagulopathy and
• disruption of the integrity of microvascular endothelium.

Asymmetrical dimethyl arginine (ADMA) is an endogenous non-selective inhibitor of nitric oxide synthase that may influence the severity of organ failure and the occurrence of shock secondary to an infectious insult. Levels may be genetically determined by a promoter polymorphism in a regulatory gene encoding dimethylarginine dimethylaminohydrolase II (DDAH II).

A prospective observational study was designed, and 47 intensive care unit (ICU) patients with severe sepsis and 10 healthy controls were enrolled. Serum ADMA and IL-6 were assayed on admission to the ICU and seven days later. Allelic variation for a polymorphism at position -449 in the DDAH II gene was assessed in each patient.
ADMA levels and Sequential Organ Failure Assessment scores were directly associated on day one (p = 0.0001) and day seven (p = 0.002). The degree of acidaemia and lactaemia was directly correlated with ADMA levels at both time points (p < 0.01). On day seven, IL-6 was directly correlated with ADMA levels (p = 0.006). The variant allele with G at position -449 in the DDAH II gene was associated with increased ADMA concentrations at both time points (p < 0.05).
Severity of organ failure, inflammation and presence of early shock in severe sepsis are associated with increased ADMA levels. ADMA concentrations may be influenced by a polymorphism in the DDAH II gene.

Several studies have added to the confusion surrounding the role of NO by demonstrating no effect of NO or NOS inhibition on the myocardium or on b-adrenergic responsiveness. Nevertheless, in most studies,

• low-to-moderate doses of iNOS inhibitors restore myocardial contractility in hearts exposed to proinflammatory cytokines, whereas
• at higher doses, the effects are reversed.

This finding may indicate that small amounts of NO produced by iNOS may be necessary to maintain contractility and can be cardio-protective in experimental sepsis.

Major cellular sources of ROS in living cells. Novo and Parola Fibrogenesis & Tissue Repair 2008 1:5 doi:10.1186/1755-1536-1-5 (Photo credit: Wikipedia)

References

1. VM Victor, K J McCreath and M Rochaa. Recent Progress in Pharmacological Research of Antioxidants in Pathological Conditions: Cardiovascular Health. Recent Patents on Anti-Infective Drug Discovery, 2006, 1, 17-31 17.
2. G Cottera, E Kaluskia, O Miloa, A Blatta, et al. LINCS: L-NAME (a NO synthase inhibitor) In the treatment of refractory Cardiogenic Shock. A prospective randomized study. The European Society of Cardiology. 2012. doi:10.1016/S0195-668X(03)00193-3 http://eurheartj.oxfordjournals.org/
3. Ve Laubach, Eg Shesely, O Smithies, Pa Sherman. Mice lacking inducible nitric oxide synthase are not resistant to lipopolysaccharide-induced death. Proc. Natl. Acad. Sci. USA 1995; 92:10688-10692, Genetics
4. NS Shah and TR Billiar. Role of Nitric Oxide in Inflammation and Tissue Injury during Endotoxemia and Hemorrhagic Shock. Environ Health Perspect 106(Suppl 5):1139-1143 (1998). http://ehpnetl.niehs.nih.gov/docs/1998/Suppl-5/1139-1 143shah/abstract.html
5. CC Wang, JE Lai, LG Chen, KY Yen, et al. Inducible Nitric Oxide Synthase Inhibitors of Chinese Herbs. Part 2: Naturally Occurring Furanocoumarins’. Bioorganic & Medicinal Chemistry 2000; 8:2701-2707.
6. MJ O’Dwyer, F Dempsey, V Crowley, DP Kelleher, R McManus, T Ryan. Septic shock is correlated with asymmetrical dimethyl arginine levels, which may be influenced by a polymorphism in the dimethylarginine dimethylaminohydrolase II gene: a prospective observational study. Critical Care 2006; 10:R139 (doi:10.1186/cc5053) http://ccforum.com/content/10/5/R139
7. C Szab, Gj Southan, And C Thiemermann. Beneficial effects and improved survival in rodent models of septic shock with S-methylisothiourea sulfate, a potent and selective inhibitor of inducible nitric oxide synthase. Proc. Natl. Acad. Sci. USA 1994; 91:12472-12476. Pharmacology
8. Wm Kazmierski, G Wolberg, Jgwilson, et al. Iron chelates bind nitric oxide and decrease mortality in an experimental model of septic shock. Proc. Natl. Acad. Sci. USA 1996;93:9138-9141.
9. L Molina, S Studenberg, G Wolberg, W Kazmierski, et al. Efficacy of Treatment With the Iron (III) Complex of Diethylenetriamine Pentaacetic Acid in Mice and Primates Inoculated With Live Lethal Dose 100 Escherichia coli. J. Clin. Invest 1996; 98(1): 192-198. 0021-9738/96/07/192/07
10. N Kayhan, B Funke, LO Conzelmann, H Winkler, et al. The adenosine deaminase inhibitor erythro-9-[2-hydroxyl-3-nonyl]-adenine decreases intestinal permeability and protects against experimental sepsis: a prospective, randomised laboratory investigation. Critical Care 2008, 12:R125 (doi:10.1186/cc7033) http://ccforum.com/content/12/5/R125
11. RD Goldfarb, A Marton, É Szabó, L Virág, et al.. Protective effect of a novel, potent inhibitor of poly(adenosine 5′-diphosphate-ribose) synthetase in a porcine model of severe bacterial sepsis. Crit Care Med 2002; 30:974 –980.
12. B Veres, F Gallyas Jr, G Varbiro, Z Berente, et al. Decrease of the inflammatory response and induction of the Akt/protein kinase B pathway by poly-(ADP-ribose) polymerase 1 inhibitor in endotoxin-induced septic shock. Biochemical Pharmacology 2003; 65: 1373–1382.
13. C Martinez, C Abad, M Delgado, A Arranz, et al. Anti-inflammatory role in septic shock of pituitary adenylate cyclase-activating polypeptide receptor. PNAS 2002; 99(2):1053–1058. doi 10.1073 pnas.012367999

• endogenously produced VIP and PACAP are participants of the natural anti-inflammatory machinery.
• VIP and PACAP are two attractive candidates for the development of therapies against acute and chronic inflammatory diseases, septic shock, and autoimmune diseases

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Nitric Oxide and Immune Responses: Part 1

Curator and Reporter: Aviral Vatsa PhD, MBBS

Based on: A review by Wink et al., 2011

This is the first part of a two part post

Nitric oxide (NO), reactive nitrogen species (RNS) and reactive oxygen species (ROS) perform dual roles as immunotoxins and immunomodulators. An incoming immune signal initiates NO and ROS production both for tackling the pathogens and modulating the downstream immune response via complex signaling pathways. The complexity of these interactions is a reflection of involvement of redox chemistry in biological setting (fig. 1)

Fig 1. Image credit: (Wink et al., 2011)

Previous studies have highlighted the role of NO in immunity. It was shown that macrophages released a substance that had antitumor and antipathogen activity and required arginine for its production (Hibbs et al., 1987, 1988). Hibbs and coworkers further strengthened the connection between immunity and NO by demonstrating that IL2 mediated immune activation increased NO levels in patients and promoted tumor eradication in mice (Hibbs et al., 1992; Yim et al., 1995).

In 1980s a number of authors showed the direct evidence that macrophages made nitrite, nitrates and nitrosamines. It was also shown that NO generated by macrophages could kill leukemia cells (Stuehr and Nathan, 1989). Collectively these studies along with others demonstrated the important role NO plays in immunity and lay the path for further research in understanding the role of redox molecules in immunity.

NO is produced by different forms of nitric oxide synthase (NOS) enzymes such as eNOS (endothelial), iNOS (inducible) and nNOS (neuronal). The constitutive forms of eNOS generally produce NO in short bursts and in calcium dependent manner. The inducible form produces NO for longer durations and is calcium independent. In immunity, iNOS plays a vital role. NO production by iNOS can occur over a wide range of concentrations from as little as nM to as much as µM. This wide range of NO concentrations provide iNOS with a unique flexibility to be functionally effective in various conditions and micro-environements and thus provide different temporal and concentration profiles of NO, that can be highly efficient in dealing with immune challenges.

Redox reactions in immune responses

NO/RNS and ROS are two categories of molecules that bring about immune regulation and ‘killing’ of pathogens. These molecules can perform independently or in combination with each other. NO reacts directly with transition metals in heme or cobalamine, with non-heme iron, or with reactive radicals (Wink and Mitchell, 1998). The last reactivity also imparts it a powerful antioxidant capability. NO can thus act directly as a powerful antioxidant and prevent injury initiated by ROS (Wink et al., 1999). On the other hand, NO does not react directly with thiols or other nucleophiles but requires activation with superoxide to generate RNS. The RNS species then cause nitrosative and oxidative stress (Wink and Mitchell, 1998).

The variety of functions achieved by NO can be understood if one looks at certain chemical concepts. NO and NO₂ are lipophilic and thus can migrate through cells, thus widening potential target profiles. ONOO-, a RNS, reacts rapidly with CO₂ that shortens its half life to <10 ms. The anionic form and short half life limits its mobility across membranes. When NO levels are higher than superoxide levels, the CO₂-OONO–intermediate is converted to NO₂ and N₂O₃ and changes the redox profile from an oxidative to a nitrosative microenvironment. The interaction of NO and ROS determines the bioavailability of NO and proximity of RNS generation to superoxide source, thus defining a reaction profile. The ROS also consumes NO to generate NO₂ and N₂O₃ as well as nitrite in certain locations. The combination of these reactions in different micro-environments provides a vast repertoire of reaction profiles for NO/RNS and ROS entities.

The Phagosome ‘cauldron’

The phagosome provides an ‘isolated’ environment for the cell to carry out foreign body ‘destruction’. ROS, NO and RNS interact to bring about redox reactions. The concentration of NO in a phagosome can depend on the kind of NOS in the vicinity and its activity and other localised cellular factors. NO and is metabolites such as nitrites and nitrates along with ROS combine forces to kill pathogens in the acidic environment of the phagosome as depicted in the figure 2 below.

Fig 2. The NO chemistry of the phagosome. (image credit: (Wink et al., 2011)

This diagram depicts the different nitrogen oxide and ROS chemistry that can occur within the phagosome to fight pathogens. The presence of NOX2 in the phagosomes serves two purposes: one is to focus the nitrite accumulation through scavenging mechanisms, and the second provides peroxide as a source of ROS or FA generation. The nitrite (NO₂−) formed in the acidic environment provides nitrosative stress with NO/NO₂/N₂O₃. The combined acidic nature and the ability to form multiple RNS and ROS within the acidic environment of the phagosome provide the immune response with multiple chemical options with which it can combat bacteria.

Bacteria

There are various ways in which NO combines forces with other molecules to bring about bacterial killing. Here are few examples

E.coli: It appears to be resistant to individual action of NO/RNS and H₂O₂ /ROS. However, when combined together, H₂O₂ plus NO mediate a dramatic, three-log increase in cytotoxicity, as opposed to 50% killing by NO alone or H₂O₂ alone. This indicates that these bacteria are highly susceptible to their synergistic action.

Staphylococcus: The combined presence of NO and peroxide in staphylococcal infections imparts protective effect. However, when these bacteria are first exposed to peroxide and then to NO there is increased toxicity. Hence a sequential exposure to superoxide/ROS and then NO is a potent tool in eradicating staphylococcal bacteria.

Mycobacterium tuberculosis: These bacterium are sensitive to NO and RNS, but in this case, NO₂ is the toxic species. A phagosome microenvironment consisting of ROS combined with acidic nitrite generates NO₂/N₂O₃/NO, which is essential for pathogen eradication by the alveolar macrophage. Overall, NO has a dual function; it participates directly in killing an organism, and/or it disarms a pathway used by that organism to elude other immune responses.

Parasites

Many human parasites have demonstrated the initiation of the immune response via the induction of iNOS, that then leads to expulsion of the parasite. The parasites include Plasmodia(malaria), Leishmania(leishmaniasis), and Toxoplasma(toxoplasmosis). Severe cases of malaria have been related with increased production of NO. High levels of NO production are however protective in these cases as NO was shown to kill the parasites (Rockett et al., 1991; Gyan et al., 1994). Leishmania is an intracellualr parasite that resides in the mamalian macrophages. NO upregulation via iNOS induction is the primary pathway involved in containing its infestation. A critical aspect of NO metabolism is that NOHA inhibits AG activity, thereby limiting the growth of parasites and bacteria including Leishmania, Trypanosoma, Schistosoma, Helicobacter, Mycobacterium, and Salmonella, and is distinct from the effects of RNS. Toxoplasma gondii is also an intracellular parasite that elicits NO mediated response. INOS knockout mice have shown more severe inflammatory lesions in the CNS that their wild type counterparts, in response to toxoplasma exposure. This indicates the CNS preventative role of iNOS in toxoplasmosis (Silva et al., 2009).

Virus

Viral replication can be checked by increased production of NO by induction of iNOS (HIV-1, coxsackievirus, influenza A and B, rhino virus, CMV, vaccinia virus, ectromelia virus, human herpesvirus-1, and human parainfluenza virus type 3) (Xu et al., 2006). NO can reduce viral load, reduce latency and reduce viral replication. One of the main mechanisms as to how NO participates in viral eradication involves the nitrosation of critical cysteines within key proteins required for viral infection, transcription, and maturation stages. For example, viral proteases or even the host caspases that contain cysteines in their active site are involved in the maturation of the virus. The nitrosative stress environment produced by iNOS may serve to protect against some viruses by inhibiting viral infectivity, replication, and maturation.

To be continued in part 2 …

Bibliography

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Further reading on NO:

Nitric Oxide in bone metabolism July 16, 2012

Author: Aviral Vatsa PhD, MBBS

http://pharmaceuticalintelligence.com/2012/07/16/nitric-oxide-in-bone-metabolism/?goback=%2Egde_4346921_member_134751669

Nitric Oxide production in Systemic sclerosis July 25, 2012

Curator: Aviral Vatsa, PhD, MBBS

http://pharmaceuticalintelligence.com/2012/07/25/nitric-oxide-production-in-systemic-sclerosis/?goback=%2Egde_4346921_member_138370383

Nitric Oxide Signalling Pathways August 22, 2012 by

Curator/ Author: Aviral Vatsa, PhD, MBBS

http://pharmaceuticalintelligence.com/2012/08/22/nitric-oxide-signalling-pathways/?goback=%2Egde_4346921_member_151245569

Nitric Oxide: a short historic perspective August 5, 2012

Author/Curator: Aviral Vatsa PhD, MBBS

http://pharmaceuticalintelligence.com/2012/08/05/nitric-oxide-a-short-historic-perspective-7/

Nitric Oxide: Chemistry and function August 10, 2012

Curator/Author: Aviral Vatsa PhD, MBBS

http://pharmaceuticalintelligence.com/2012/08/10/nitric-oxide-chemistry-and-function/?goback=%2Egde_4346921_member_145137865

Nitric Oxide and Platelet Aggregation August 16, 2012 by

Author: Dr. Venkat S. Karra, Ph.D.

http://pharmaceuticalintelligence.com/2012/08/16/no-and-platelet-aggregation/?goback=%2Egde_4346921_member_147475405

The rationale and use of inhaled NO in Pulmonary Artery Hypertension and Right Sided Heart Failure August 20, 2012

Author: Larry Bernstein, MD

http://pharmaceuticalintelligence.com/2012/08/20/the-rationale-and-use-of-inhaled-no-in-pulmonary-artery-hypertension-and-right-sided-heart-failure/

Nitric Oxide: The Nobel Prize in Physiology or Medicine 1998 Robert F. Furchgott, Louis J. Ignarro, Ferid Murad August 16, 2012

Reporter: Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2012/08/16/nitric-oxide-the-nobel-prize-in-physiology-or-medicine-1998-robert-f-furchgott-louis-j-ignarro-ferid-murad/

Coronary Artery Disease – Medical Devices Solutions: From First-In-Man Stent Implantation, via Medical Ethical Dilemmas to Drug Eluting Stents August 13, 2012

Author: Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2012/08/13/coronary-artery-disease-medical-devices-solutions-from-first-in-man-stent-implantation-via-medical-ethical-dilemmas-to-drug-eluting-stents/

Nano-particles as Synthetic Platelets to Stop Internal Bleeding Resulting from Trauma

August 22, 2012

Reported by: Dr. V. S. Karra, Ph.D.

http://pharmaceuticalintelligence.com/2012/08/22/nano-particles-as-synthetic-platelets-to-stop-internal-bleeding-resulting-from-trauma/

Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production July 19, 2012

Curator and Research Study Originator: Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2012/07/19/cardiovascular-disease-cvd-and-the-role-of-agent-alternatives-in-endothelial-nitric-oxide-synthase-enos-activation-and-nitric-oxide-production/

Macrovascular Disease – Therapeutic Potential of cEPCs: Reduction Methods for CV Risk

July 2, 2012

An Investigation of the Potential of circulating Endothelial Progenitor Cells (cEPCs) as a Therapeutic Target for Pharmacological Therapy Design for Cardiovascular Risk Reduction: A New Multimarker Biomarker Discovery

Curator: Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2012/07/02/macrovascular-disease-therapeutic-potential-of-cepcs-reduction-methods-for-cv-risk/

Bone remodelling in a nutshell June 22, 2012

Author: Aviral Vatsa, Ph.D., MBBS

http://pharmaceuticalintelligence.com/2012/06/22/bone-remodelling-in-a-nutshell/

Targeted delivery of therapeutics to bone and connective tissues: current status and challenges- Part, September  

Author: Aviral Vatsa, PhD, September 23, 2012

http://pharmaceuticalintelligence.com/2012/09/23/targeted-delivery-of-therapeutics-to-bone-and-connective-tissues-current-status-and-challenges-part-i/

Calcium dependent NOS induction by sex hormones: Estrogen

Curator: S. Saha, PhD, October 3, 2012

http://pharmaceuticalintelligence.com/2012/10/03/calcium-dependent-nos-induction-by-sex-hormones/

Nitric Oxide and Platelet Aggregation,

Author V. Karra, PhD, August 16, 2012

http://pharmaceuticalintelligence.com/2012/08/16/no-and-platelet-aggregation/

Bystolic’s generic Nebivolol – positive effect on circulating Endothelial Progenitor Cells endogenous augmentation

Curator: Aviva Lev-Ari, PhD, July 16, 2012

http://pharmaceuticalintelligence.com/?s=Nebivolol

Endothelin Receptors in Cardiovascular Diseases: The Role of eNOS Stimulation

Author: Aviva Lev-Ari, PhD, 10/4/2012

http://pharmaceuticalintelligence.com/2012/10/04/endothelin-receptors-in-cardiovascular-diseases-the-role-of-enos-stimulation/

Inhibition of ET-1, ETA and ETA-ETB, Induction of NO production, stimulation of eNOS and Treatment Regime with PPAR-gamma agonists (TZD): cEPCs Endogenous Augmentation for Cardiovascular Risk Reduction – A Bibliography

Curator: Aviva Lev-Ari, 10/4/2012.

http://pharmaceuticalintelligence.com/2012/10/04/inhibition-of-et-1-eta-and-eta-etb-induction-of-no-production-and-stimulation-of-enos-and-treatment-regime-with-ppar-gamma-agonists-tzd-cepcs-endogenous-augmentation-for-cardiovascular-risk-reduc/

Nitric Oxide Nutritional remedies for hypertension and atherosclerosis. It’s 12 am: do you know where your electrons are?

Author and Reporter: Meg Baker, 10/7/2012.

http://pharmaceuticalintelligence.com/2012/10/07/no-nutritional-remedies-for-hypertension-and-atherosclerosis-its-12-am-do-you-know-where-your-electrons-are/

 

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