An Overview of the Automated and On-Line Systems to Assess the Oxidative Potential of Particulate Matter

Recent years have seen a significant increase in the scientific literature related to various methods for analyzing oxidative potential (OP) of atmospheric particulate matter (PM). The presence of several types of PM, differing chemical and physical properties, released by both anthropogenic and natural sources, leads to numerous health issues in living organisms and represents an attractive target for air quality monitoring. Therefore, several studies have focused on developing rapid and self-operative tests, employing different target molecules to assess OP of atmospheric aerosols as well as unique approaches to overcome some of the most common laboratory-related issues in this kind of analysis. This work provides an overview of online and automated systems, as well as a broad picture of the state-of-art of the various devices and methods developed on this topic over the last two decades. Moreover, representative studies on this subject will be discussed, analyzing the advantages and drawbacks of the developed automated techniques

Novel Methods to Characterise Atmospherically Relevant Organic Radicals and Reactive Oxygen Species

A key reaction in the troposphere involves the oxidation of biogenic and anthropogenic alkenes with ozone, which contributes to local photochemical smog. It is generally accepted that this reaction proceeds via a reactive intermediate often called the Criegee intermediate (CI). This reaction is known to produce a plethora of oxidised organic compounds, which contribute to ozone formation and secondary organic aerosol production, two of the main characteristics of a polluted atmosphere. Furthermore, epidemiological studies have shown a close correlation between exposure to ambient organic aerosol and adverse human health effects. The toxicological mechanisms leading to this observation are still poorly characterised, although studies suggest that reactive oxygen species present in organic aerosol are a major contributor. Reactive oxygen species and reactive intermediates represent a large uncertainty in tropospheric chemistry, and pose an analytical challenge due to their high reactivity and typically low concentrations. This emphasises the need for the development of new methods to characterise the chemistry of these species. In this thesis, several novel laboratory based techniques have been developed in order to characterise and quantify reactive intermediates and reactive oxygen species. New methods to facilitate the detection of CIs in both the gas and particle phase are presented. Spin trap molecules are used to scavenge CIs to form stable 1:1 adducts which are subsequently detected and quantified using mass spectrometry. The chemistry of CIs with spin traps is extensively investigated. The unique capability of this technique to simultaneously characterise multiple CIs generated from a variety of atmospherically relevant organic precursors in the gas phase is demonstrated. This technique was further developed to facilitate the detection of CIs in secondary organic aerosol, representing the development of a method capable of characterising low volatility CIs and other reactive intermediates in the condensed phase. Furthermore, two new chemical fluorescence assays have been developed to quantify both organic radicals and reactive oxygen species in organic aerosol. A novel profluorescent spin trap assay was applied to quantify radical concentrations in organic aerosol. A series of experiments were then devised to investigate the lifetime of organic radicals in secondary organic aerosol. A second assay, based on physiologically relevant ascorbic acid chemistry, was also developed to measure the concentrations of toxicologically relevant reactive oxygen species in secondary organic aerosol. The quantitative capability of this assay was extensively characterised. The assay was incorporated into a prototype instrument capable of measuring particle-bound reactive oxygen species on-line, and the assays’ sensitivity to secondary organic aerosol was demonstrated

Developing an instrument to quantify aerosol toxicity

Large-scale epidemiological studies have consistently shown that exposure to ambient particulate matter (PM) is responsible for a variety of adverse health effects. However, the specific physical and chemical properties of particles that are responsible for observed health effects, as well as the underlying mechanisms of particle toxicity upon exposure, remain largely uncertain. Studies have widely suggested that the oxidative potential (OP) of aerosol particles is a key metric to quantify particle toxicity. OP is defined as the ability of aerosol particle components to produce reactive oxidative species (ROS) and deplete antioxidants in vivo. Traditional methods for measuring OP using acellular assays largely rely on analyzing PM collected in filters offline. This is labor intensive and involves a substantial time delay between particle collection and OP analysis. It therefore likely underestimates particle OP, because many reactive chemical components which are contributing to OP are short-lived and therefore degrade prior to offline analysis. We investigated these differences in online and offline measurements with different acellular assays and with cellular methods and could show that for biogenic secondary organic aerosol (SOA), a large fraction decays within minutes to hours. Thus, new techniques are required to provide a robust and rapid quantification of particle OP, capturing the chemistry of oxidizing and short-lived highly reactive aerosol components and their concentration dynamics in the atmosphere. To address these measurement shortcomings, we developed a portable online instrument that directly samples particles into an ascorbic acid-based assay under physiologically relevant conditions of pH 6.8 and 37 °C, providing continuous accurate OP measurements with a high time resolution (5 min). This online oxidative potential ascorbic acid instrument (OOPAAI) runs autonomously for up to three days and has a detection limit of about 5 μg/m3 in an urban environ- ment, which allows the characterization of particle OP, even in low-pollution areas. With this novel instrument, we not only measured ambient aerosol, but also conducted various laboratory campaigns where we investigated the toxicity of various aerosol systems. Primary and secondary emissions with different aging times from car exhaust were measured and compared to primary and secondary aerosols from residential wood combustion, showing a higher toxicity for residential wood combustion for primary and secondary aerosols. Furthermore, we investigated the influence of transition metals like copper and iron on the OP of secondary organic aerosol. We could show that there is a synergistic effect for biogenic SOA with copper and for anthropogenic SOA with copper and iron, but an antagonistic effect with iron and biogenic SOA measured with the OOPAAI

Evaluation of the effects of PM emitted by specific emission sources on environment and health

The health burden due to particulate matter (PM) air pollution (PM10 and PM2.5) is one of the biggest environmental health concerns in the WHO European Region and around the world. A particular challenge in this research field is about the identification of the physical and chemical characteristics of PM able to reveal the correlation that links fine particle pollutants and respiratory morbidity and mortality. PM is a complex, heterogeneous mixture, whose chemical and physical characteristics (particle size distribution, chemical composition) changes in time and space and depends on various factors (sources, atmospheric chemistry and weather conditions). In literature, most studies associate PM emitted by the major combustion sources, mobile and stationary, with a range of serious health effects, including increased morbidity and mortality from cardiovascular and respiratory conditions. Current knowledge, however, does not allow a quantification of the health effects of PM emissions from different sources or from individual PM components. Therefore, the study of a depth chemical characterization of the individual emissive sources would be helpful in identification of possible PM toxic effects. Another important factor in the assessment of the aspects that link the human health to particulate pollution is the size of the particle to which the population is exposed. In fact, the size of the particles, together with their chemical composition, are fundamental indicator of health risk. Particle size determines in which region of the respiratory tract particles are deposited, as well as the amount of particles deposited. In this optic, a better understanding of the strength of individual emission sources, of the size of the emitted particles and of their chemical composition could facilitate the design of targeted abatement policies more effective to reduce the burden of diseases due to air pollution. My PhD work was carried out in response to all the above-mentioned needs. In fact, during these three years, I have been studying and deepened all those factors (size, emission sources, chemical composition and indicators of oxidative stress) that could be crucial, to the scientific world, for a better understanding of the PM's harmful effects both on humans and on environment. I have been involved in the study of possible techniques able to monitor and characterize as well as possible, the different emission sources, some of which can only be identified by using high timeresolved methods. I have been also involved in the study of different methods that can quantify the capacity of inhaled PM to cause oxidative stress within the lung, which seems to be one of the main mechanisms for the adverse cardio-respiratory health effects observed in epidemiological studies

Renewable diesel fuels and emission control strategies : Implications for occupational exposure, human health, and the environment

Combustion of fossil diesel is a major environmental problem for both the climate and human health. Renewable diesel fuels have been developed and introduced to the market to reduce the net CO2 emissions. Emission control strategies, such as aftertreatment systems, have been implemented to reduce the health hazardous particulate matter (PM) and nitrogen oxides (NOx) emissions. The overall aim of this thesis was to understand the effect of introducing renewable diesel fuels and emission abatement techniques on health relevant exhaust emissions. Laboratory studies were performed to assess the primary and secondary emissions from a heavy-duty diesel engine fueled by the renewable diesel fuels HVO (hydrotreated vegetable oil) and RME (rapeseed methyl ester).The emissions were characterized by detailed particle and gas measurements. We also evaluated the effect of using an aftertreatment system consisting of a diesel oxidation catalyst (DOC) and a diesel particle filter (DPF) on the exhaust emissions. The occupational exposure to diesel exhaust from vehicles in a Swedish modern underground mine was quantified and evaluated in relation to the vehicles’ level of emission reduction technology. The underground ambient concentrations were quantified, and real-world emission factors were calculated. The short-term health effects of HVO exhaust from modern non-road vehicles (2019), with or without the PM fraction, were investigated in a controlled human exposure chamber study. Replacing fossil diesel with HVO and RME significantly reduced the PM emissions, especially the soot emissions(measured as elemental carbon [EC] and equivalent black carbon [eBC]). The fuel change also reduced the hydrocarbon and carbon monoxide emissions, particularly from RME. The significantly reduced hydrocarbon emissions from RME also reduced the secondary aerosol formation, and thus potentially reducing the total atmospheric particle mass burden. Aftertreatment systems containing both a DOC and DPF were very efficient in removing the particle concentrations in the laboratory studies for all fuels. However, as long as a large portion of the vehicle fleet does not have any PM removal systems, the usage of HVO and RME will have a positive impact on overall PM reductions. The average occupational exposure concentration of EC was 7 μg m-3 in the underground mine. This is much lower than the future EU occupational exposure limit (OEL) for diesel exhaust (50 μg EC m-3, from 2026underground). However, epidemiological studies suggest health-based limits closer to 1 μg m-3, which indicates that we should aim to further reduce the exposure. The measured EC exposures ware reduced in areas where vehicles had DPFs. Short-term exposure to HVO exhaust below the EU OELs did not cause severe pulmonary function changes in healthy subjects. However, the subjects experienced an increase in self-rated mild irritation symptoms, and a mild decrease in nasal patency after both the particle-laden and the particle-free HVO exposure. This may indicate irritative effects from exposure to HVO exhaust from modern non-road vehicles below future OELs. Air pollution from combustion sources (not only from vehicles) is a global problem that will be present for years to come. Due to the many adverse effects linked to aerosol air pollution, measures need to be taken to reduce the particle exposures in environmental and occupational settings. The future occupational exposure limit of 50 μg ECm-3 is still much higher than proposed health-based limits. For combustion vehicles, the most efficient way to reduce EC emissions is by using aftertreatment systems focused on removing the PM, such as DPFs. Resources need to be focused on ensuring that such systems are in place and working effectively in all combustion vehicles. This is especially the case in highly exposed areas such as in cities and enclosed work environments

Examination of the Adverse Effects of Exposure to Gaseous and Particulate Oxidant Air Pollutants in Human Airway Epithelial Cells

Human exposure to ambient air pollution is a pervasive global public health problem. Ambient levels of air pollutants, such as particulate matter and ozone, are associated with multiple adverse health effects, including increases in the incidence of morbidity and mortality. The underlying mechanism(s) responsible for the adverse effects of most air pollutants is not well understood. However, oxidative stress has been implicated as being a major contributor to the mechanism of toxic action of numerous gaseous and particulate air pollutants. The lungs serve as the primary route of exposure for air pollutants, making cells of the respiratory epithelia principal targets for many of the toxicological outcomes of air pollution exposure. The concentrations of gaseous and particulate matter (PM) air pollutants are primary determinants of the pulmonary toxicity resultant from air pollutant exposure. The study of oxidative responses to air pollutant exposure requires that a number of methodological challenges be overcome. The studies of this dissertation purposely address these challenges in the following manner: 1) Development and implementation of imaging methodologies for the investigation of effects resulting from particulate and gaseous air pollutant exposure to Human Airway Epithelial Cells (HAEC); 2) Examination of the cellular mechanisms that underlie oxidative stress responses to air pollution exposures in HAEC using live cell imaging methodologies; and 3) Examination of factors that mediate air pollution-induced changes in intracellular redox status. The major features of this body of work were able to validate and establish significant methodologies for examining the interaction of nano-scaled particulates with cellular environments, and observe oxidative alterations in the intracellular redox environment of oxidant-exposed cells in real-time. Moreover, these findings reveal that exposure to oxidative air pollutants, such as ozone, induces a profound increase in the intracellular glutathione redox potential of human airway epithelial cells that is indicative of an oxidant-dependent impairment of redox homeostasis in the cell. Cumulatively, this work advances current toxicological knowledge regarding the spatiotemporal interaction of gaseous and particulate air pollutants with cellular environments, while producing effective methodologies for the assessment of implications resulting from air pollutant exposure. Furthermore, the methodologies described herein can be used in broader toxicological applications assessing similar endpoints from other types of xenobiotic exposures.Doctor of Philosoph

Synthesis, endogenous detection, and mitochondrial function of the hydroxy-substituted Coenzyme Q10 derivative HO-Q10

Quinones are redox-active molecules playing important roles in all organisms. In humans, the para-benzoquinone derivative Coenzyme Q10 (CoQ10) carrying a chain of 10 isoprene units and two neighbouring methoxy groups at the benzoquinone ring is found ubiquitously. It is an essential electron and proton transporter in the mitochondrial respiratory chain and fulfils various important functions like regulating redox homeostasis and membrane viscosity. In 2011, Bogeski et al. chemically modified the functional head group of CoQ10 exchanging one methoxy group by a hydroxy group. The hydroxy analogue can also be produced by CYP450 present in mitochondria and endoplasmic reticulum and is a postulated intermediate in CoQ biosynthesis that had not been detected in eukaryotes. The aim of this thesis was to gain first insights into the biological role of the mono-demethylated Coenzyme Q10. Therefore, within this study and my precedent master thesis, the CoQ10 derivative, HO-CoQ10, was synthesized and purified in sufficient amounts for the first time. Its structure could be verified using mass spectrometry and 1H/13C 2-dimensional nuclear magnetic resonance spectroscopy. The synthesis was confirmed to always produce a constitutional isomer mixture of HO-CoQ10 modified at the 2- or 3-position of the quinone ring. Due to the high lipophilicity mediated by the long isoprene chain, reliable transition to the aqueous phase was crucial for experiments. An ethanolic solution of 1 mM CoQ10 and 5 mM HO-CoQ10 was stable at room temperature and could be diluted in aqueous media. Hence, most experiments were conducted with 1% ethanol resulting in a maximal concentration of 10 μM CoQ10. To understand the physiological importance of HO-CoQ10, its endogenous occurrence was clarified using ultra-high-pressure liquid chromatography coupled to tandem mass spectrometry. In isopropanol extracts from crude bovine heart mitochondria, HO-CoQ10 was detected for the first time and estimated to have a concentration of 100 μM in mitochondrial membranes. Evaluating toxicity of exogenously applied HO-CoQ10, no effect on cancer cell lines cultivated under standard cell culture conditions was found: No interference with metabolic activity and proliferation was observed in HeLa, MelJuso and Jurkat T cells using the CellTiter-Blue® reduction assay and apoptosis was not induced in Jurkat T cells analysing caspase activity using Caper-GR sensor. Detecting HO-CoQ10 in mitochondria and considering the essential role of CoQ10 in the electron transport chain, its influence on respiration was evaluated measuring oxygen consumption of isolated cardiac mitochondria from BL6N mice using a Clark-type electrode. HO-CoQ10 inhibited Complex I-, II-, and III-linked respiration. Surprisingly, also the native substance CoQ10 intervened with Complex I- and II-linked respiration, but to a lower extent than HO-CoQ10. Extramitochondrial calcium enhanced inhibition via CoQ10 and HO-CoQ10 by the same factor. Since respiration buffers contained inorganic phosphate affecting free metal concentrations, free calcium was defined using the fluorescent calcium indicator fura-2. Photometric activity assays of respiratory chain enzymes from bovine heart mitochondria showed that HO-CoQ10 but not CoQ10 inhibited both oxidoreductase activity of Complex I and II. Decyl-ubiquinol:cytochrome c oxidoreductase (Complex III) was unaffected. In-gel activity staining of Complex I and II did not show interference on oxidase activities indicating that HO-CoQ10 acts on Q-binding sites of the complexes. Since CoQ10 is an important antioxidant in vivo but also shows prooxidant activity under distinct circumstances depending on its redox state, H2O2 production in the supernatant of coupled mouse heart mitochondria was examined using an HRP-based assay with Amplex® UltraRed. HO-CoQ10 increased Complex I- and II-linked ROS (reactive oxygen species) formation rate. CI-linked rates were similar for HO-CoQ10 and the ubiquinone- binding site inhibitor rotenone. In contrast, production rates induced by the Qi-site inhibitor of Complex III, antimycin A, were considerably higher suggesting a distinct mechanism of ROS formation. Glycerol dehydrogenase-linked H2O2 production was increased by antimycin A and slightly reduced by HO-CoQ10. To assess the correlation of respiration and ROS production, oxygen consumption and superoxide production were detected simultaneously by electron spin resonance spectroscopy with the oxygen sensor trityl and the spin probe for superoxide CMH. Similar to rotenone, CM• formation rate was not influenced by HO-CoQ10. Electron distribution in Complex I-associated iron-sulphur clusters of NADH-energized bovine heart mitochondria, visualised with low-temperature electron spin resonance spectroscopy, was not altered by HO- CoQ10. This work provides the first insight into the biological significance of the neglected hydroxy analogue of CoQ10. Focussing on mitochondrial respiration, the most studied and biologically important role of Coenzyme Q, it was found that both CoQ10 and HO-CoQ10 inhibit respiration. Enrichment of the substances was shown to decrease membrane fluidity in other studies, which might be the underlying mechanism diminishing electron transfer efficiency. Calcium potentiating the inhibition by both substances supports the observation that HO-CoQ10 as well as CoQ10 are able to bind calcium. Due to its lower redox potential, HO-CoQ10 has been predicted to be a more potent antioxidant with a potential to substitute CoQ10 in the medication of ROS related diseases. However, it was found to inhibit activity of respiratory chain complexes and stimulated ROS production most likely via blocking Q-binding sites. Downregulation of respiration and stimulation of ROS production by HO-CoQ10, found in small portions in metabolic active heart tissue, suggests a role in regulation of energy metabolism and might act as an internal brake for growth when synthesis is upregulated.Synthese, endogene Detektion und mitochondriale Funktion des hydroxy-substituierten Coenzym Q10-Derivats HO-CoQ10: Chinone sind redoxaktive Moleküle, die in allen Organismen wichtige Aufgaben erfüllen. Im Menschen ist das para-Benzochinon-Derivat Coenzym Q10 (CoQ10) ubiquitär vorhanden. Am Chinonring trägt es zwei benachbarte Methoxygruppen und eine Seitekette aus zehn Isopren-Einheiten. CoQ10 ist ein essentieller Elektronen- und Protonentransporter der mitochondrialen Atmungskette und an anderen wichtigen Funktionen wie der Regulation der Redoxhomoöstase und Membranviskosität beteiligt. 2011 haben Bogeski et al. seine funktionelle Kopfgruppe chemisch modifiziert, indem sie eine der Methoxygruppen durch eine Hydroxygruppe substituiert haben. Das Hydroxy-Analogon kann auch durch CYP450-Enzyme, welche in Mitochondrien und endoplasmatischem Retikulum exprimiert sind, produziert werden und ist eine postulierte biosysnthetische Vorstufe von Coenzyme Q, die jedoch in Eukaryoten bisher nicht nachgewiesen werden konnte. In der vorliegenden Arbeit wurde erstmals die biologische Funktion von mono-demethyliertem Coenzym Q10 untersucht. Hierzu wurde das Coenzym Q10-Derivat, HO-CoQ10, im Rahmen dieser Doktorarbeit und der vorangehenden Masterarbeit synthetisiert und in ausreichender Menge aufgereinigt. Die Struktur wurde mittels 1H/13C 2-dimensionaler Kernspinresonanzspektroskopie verifiziert. Das Produkt besteht aus einer Mischung an Konstitutionsisomeren, welche die Hydroxygruppe an Position 2 oder 3 des Chinonrings tragen. Aufgrund der langen Isoprenkette handelt es sich um stark liphophile Substanzen, deren Übergang ins Wässrige für die folgenden Untersuchungen essentiell war. 1 mM CoQ10 und 5 mM HO-CoQ10 in ethanolischer Lösung waren bei Raumtemperatur stabil und konnten in wässrigen Medien verdünnt werden. Deshalb wurden die meisten Experimente mit 1 % Ethanol und einer maximalen CoQ10 Konzentration von 10 μM durchgeführt. Um die physiologische Bedeutung von HO-CoQ10 zu erfassen, wurde sein endogenes Vorkommen mittels Ultra-Hochdruckflüssigkeitschromatographie-gekoppelter Massenspektrometrie aufgeklärt. In Isopropanol- Extrakten aus unaufbereitetetn Rinderherzmitochondrien wurde HO-CoQ10 erstmals nachgewiesen und seine Konzentration in der Mitochondrienmembran auf 100 μM geschätzt. Die Toxizität von exogen appliziertem HO-CoQ10 auf unter Standard-Zellkulturbedingungen kultivierten Krebszelllinien wurde untersucht: Die metabolische Aktivität und Proliferation von HeLa, MelJuso und Jurkat T-Zellen, bestimmt mit Hilfe des CellTiter-Blue®-Reduktionsassays, war unbeeinträchtigt. Zudem konnte in Jurkat T-Zellen bei Analyse der Caspase-Aktivität mittels des Casper-GR-Sensors keine Apoptoseinduktion beobachtet werden. Da HO-CoQ10 in Mitochondrien detektiert wurde und CoQ10 eine essentielle Rolle in der Elektronentransportkette einnimmt, wurde der Einfluss auf die Respiration untersucht. Hierfür wurde der Sauerstoffverbrauch isolierter Herzmitochondrien aus BL6N-Mäusen mit Hilfe einer Clark-Elektrode gemessen. HO-CoQ10 inhibierte Complex I-, II- und III-assoziierte Atmung. Erstaunlicherweise inhibierte auch die native Substanz CoQ10 die Complex I- und II-assoziierte Respiration; dies jedoch in einem geringeren Ausmaß als HO-CoQ10. Extramitochondriales Calcium steigerte die durch CoQ10 und HO-CoQ10 vermittelte Inhibition gleichermaßen. Die Puffer für Atmungsmessungen enthalten anorganisches Phosphat, welches sich auf die Konzentration an freien Metallionen auswirkt. Deswegen musste die freie Calicumkonzentration mittels des fluoreszenten Calciumindikators Fura-2 festgelegt und -gestellt werden. Photometrische Aktivitätsbestimmungen der Atmungskettenenzyme aus Rinderherzmitochondrien zeigten, dass HO-CoQ10, jedoch nicht CoQ10 Complex I- und Complex II-Oxidoreduktase-Aktivität inhibiert. Decyl-ubichinol:Cytochrom c oxidoreduktase (Complex III) war unbeeinträchtigt. In-Gel-Aktivitätsfärbungen stellten heraus, dass HO- CoQ10 keinen Einfluss auf die Oxidase-Aktivität von Complex I und Complex II hat. Dies deutet darauf hin, dass HO-CoQ10 auf die Q-Bindestellen wirkt. Da CoQ10 ein in-vivo wichtiges Antioxidans ist, das unter bestimmten Umständen abhängig vom Redoxzustand prooxidativ wirkt, wurde die H2O2-Produktion im Überstand gekoppelter Mausherzmitochondrien mit Hilfe eines HRP-basierten Assays mit Amplex® UltraRed untersucht. HO-CoQ10 erhöhte die Complex I- und Complex II-assoziierte Produktionsrate an reaktiven Sauerstoffspezies (ROS). CI-assoziierte Raten mit HO-CoQ10 waren vergleichbar mit Raten in Anwesenheit des Ubichinon- Bindestellen-Inhibitors Rotenon. Im Gegensatz dazu zeigten Experimente mit dem Qi-Bindestellen-Inhibitor von Complex III, Antimycin A, deutlich höhere Produktionsraten und weisen damit auf einen anderen Mechanismus der ROS-Produktion hin. Glyceroldehydrogenase-assoziierte H2O2-Produktion war durch Antimycin A erhöht und durch HO-CoQ10 leicht reduziert. Um den Zusammenhang von Respiration und ROS- Produktion zu untersuchen, wurden Sauerstoffverbrauch und Superoxidproduktion simultan mitttels Elektronenspinresonsanzspektroskopie mit dem Sauerstoffsensor Trityl und der Superoxid-Spinsonde CMH gemessen. Vergleichbar mit Rotenon wurde die CM•-Bildungsrate auch von HO-CoQ10 nicht beeinflusst. Die Elektronenverteilung in Eisen-Schwefel-Clustern von Complex I in NADH-energetisierten Rinderherzmitochondrien wurde mittels Tieftemperatur-Elektronenspinresonanzspektroskopie visualisiert und war nicht durch HO-CoQ10 verändert. Die vorliegende Arbeit gibt erste Hinweise auf die biologische Bedeutung des wenig beachteten Hydroxy-Analogons von CoQ10. Es konnte gezeigt werden, dass sowohl CoQ10 als auch HO-CoQ10 die mitochondriale Atmung inhibieren. Da andere Studien zeigten, dass die Anreicherung der Substanzen die Fluidität der Membran reduziert, könnte dies der zugrunde liegende Mechanismus für eine Abnahme der Elektronentransfereffizienz sein. Die Potenzierung der Inhibition durch Calcium unterstützt die Beobachtung, dass HO-CoQ10 und CoQ10 die Fähigkeit besitzen Calcium zu binden. Aufgrund des niedrigeren Redoxpotentials wurde prognostiziert, dass HO-CoQ10 ein effektiveres Antioxidans ist und das Potential besitzt, CoQ10 in der medizinischen Behandlung von ROS-korrelierten Krankheiten zu ersetzen. Allerdings zeigte sich, dass HO-CoQ10, höchstwahrscheinlich durch die Blockade von Q-Bindestellen, Atmungskettenkomplexe hemmt und ROS-Produktion stimuliert. Herunterregulation der Respiration und Stimulation von ROS-Produktion durch HO-CoQ10, das in stoffwechselaktivem Herzgewebe in kleinen Mengen gefunden wurde, weist auf Beteiligung in der Regulation von Energiestoffwechsel hin und könnte bei Hochregulation der Synthese als interne Wachstumsbremse agieren

SUMOylation of NaV1.2 channels mediates the early response to acute hypoxia in central neurons.

The mechanism for the earliest response of central neurons to hypoxia-an increase in voltage-gated sodium current (INa)-has been unknown. Here, we show that hypoxia activates the Small Ubiquitin-like Modifier (SUMO) pathway in rat cerebellar granule neurons (CGN) and that SUMOylation of NaV1.2 channels increases INa. The time-course for SUMOylation of single NaV1.2 channels at the cell surface and changes in INa coincide, and both are prevented by mutation of NaV1.2-Lys38 or application of a deSUMOylating enzyme. Within 40 s, hypoxia-induced linkage of SUMO1 to the channels is complete, shifting the voltage-dependence of channel activation so that depolarizing steps evoke larger sodium currents. Given the recognized role of INa in hypoxic brain damage, the SUMO pathway and NaV1.2 are identified as potential targets for neuroprotective interventions

Mechanisms of augmented coronary artery constriction following exposure to diesel exhaust

Numerous epidemiology studies demonstrate that acute increases in air pollutants correlate with an increase in cardiovascular disease-related mortality. The pollutant diesel exhaust (DE) has been shown to impair both flow-mediated and agonist-induced dilation of the brachial artery, used as a surrogate for coronary artery function. It is speculated that enhanced sensitivity to the endogenous vasoconstrictor ET-1 impairs cardiac blood flow and contributes to the immediate onset of myocardial ischemia and infarction in humans following DE exposure. In addition, impaired endothelium-dependent dilation can be improved with the restoration of nitric oxide (NO) synthase (NOS) activity. We therefore sought to determine the mechanism by which inhalation of DE impairs coronary artery function by assessing responses to ET-1 and to the endothelium-dependent vasodilator acetylcholine (ACh) in arteries from rats exposed to DE compared to responses in arteries from rats exposed to filtered air. Given that DE is a source of reactive oxygen species (ROS) we hypothesized that inhaled DE generates ROS which uncouples NOS-dependent dilation to augment coronary artery constriction. We observed augmented vasoconstrictor sensitivity to ET-1 and blunted vasodilator response to ACh in coronary arteries following DE exposure. Interestingly, these alterations in vascular reactivity appear to result not only from the loss of NO, but also from a gain in NOS-derived constrictors. Furthermore, basal activity of NOS was not altered by DE exposure. Elevated ROS are known to oxidize and deplete tetrahydrobiopterin (BH4) a necessary cofactor that prevents the uncoupling of NOS. ROS scavenging or BH4 supplementation prevented the generation of superoxide in isolated arteries as did NOS inhibition. These treatments also restored dilation to ACh. Therefore, acute inhalation of DE appears to deplete bioavailable BH4, uncouple NOS and lead to NOS-dependent superoxide generation. The increased oxidative stress likely scavenges and decreases synthesis of NO leading to endothelial dysfunction which may contribute to the acute coronary events initiated by air pollution