air-sampled-filter-analysis-for-endotoxins-and-dna-content

Air-sampled Filter Analysis for Endotoxins and DNA Content

Outdoor aerosol research commonly uses particulate matter sampled on filters. This procedure enables various characterizations of the collected particles ...

Weizmann Institute of Science

Caloric restriction (CR) is currently the only therapeutic intervention known to attenuate aging in mammals, but the underlying mechanisms of this phenomenon are still poorly understood. To get more insight into these mechanisms, we took advantage of the alphaMUPA transgenic mice that previously were reported to spontaneously eat less and live longer compared with their wild-type control mice. Currently, two transgenic lines that eat less are available, thus implicating the transgenic enzyme, i.e. the urokinase-type plasminogen activator (uPA), in causing the reduced appetite. This phenotypic change could have resulted from the ectopic transgenic expression that we detected in the adult alphaMUPA brain, or alternatively, from a transgenic interference in brain development. Here, we have summarized similarities and differences so far found between alphaMUPA and calorically restricted mice. Recently, we noted several changes in the alphaMUPA liver, at the mitochondrial and cellular level, which consistently pointed to an enhanced capacity to induce apoptosis. In addition, alphaMUPA mice showed a reduced level of serum IGF-1 and a reduced incidence of spontaneously occurring or carcinogen-induced tumors in several tissues. In contrast, alphaMUPA did not differ from wild type mice in the levels of low molecular weight antioxidants when compared in several tissues at a young or an old age. Overall, the alphaMUPA model suggests that fine-tuning of the threshold for apoptosis, possibly linked in part to modulation of serum IGF-1 and mitochondrial functions, could play a role in the attenuation of aging in calorically restricted mice.

AlphaMUPA mice: a transgenic model for longevity induced by caloric restriction.

Calorie restriction (CR) extends the life span of various species through mechanisms that are as yet unclear. Recently, we have reported that mitochondrion-mediated apoptosis was enhanced in alphaMUPA transgenic mice that spontaneously eat less and live longer compared with their wild-type (WT) control mice. To understand the molecular mechanisms underlying the increased apoptosis, we compared alphaMUPA and WT mice for parameters associated with SOD2 (MnSOD), a mitochondrial antioxidant enzyme that converts superoxide radicals into H(2)O(2) and is also known to inhibit apoptosis. The SOD2-related parameters included the levels of SOD2 mRNA, immunoreactivity and enzymatic activity in the liver, lipid oxidation and aconitase activity in isolated liver mitochondria, and the sensitivity of the mice to paraquat, an agent that elicits oxidative stress. In addition, we compared the mice for the levels of SOD2 mRNA after treatment with bacterial lipopolysaccharides (LPS), and for the DNA binding activity of NFkappaB as a marker for the inflammatory state. We extended SOD2 determination to the colon, where we also examined the formation of pre-neoplastic aberrant crypt foci (ACF) following treatment with dimethylhydrazine (DMH), a colonic organotypic carcinogen. Overall, alphaMUPA mice showed reduced basal levels of SOD2 gene expression and activity concomitantly with reduced lipid oxidation, increased aconitase activity and enhanced paraquat sensitivity, while maintaining the capacity to produce high levels of SOD2 in response to the inflammatory stimulus. alphaMUPA mice also showed increased resistance to DMH-induced pre-neoplasia. Collectively, these data are consistent with a model, in which an optimal fine-tuning of SOD2 throughout a long-term regimen of reduced eating could contribute to longevity, at least in the alphaMUPA mice.

Long-lived alphaMUPA transgenic mice show reduced SOD2 expression, enhanced apoptosis and reduced susceptibility to the carcinogen dimethylhydrazine.

Manganese superoxide dismutase (MnSOD, SOD2) is an essential primary antioxidant enzyme which converts superoxide radical to hydrogen peroxide within the mitochondrial matrix. MnSOD plays a prominent role in protection against many apoptotic stimuli. Its absence may therefore impair the cellular redox balance and enhance apoptosis. Our data show that in Jurkat T cells, following oligomerization of the Fas receptor, MnSOD is selectively degraded during apoptosis. In the presence of cycloheximide, an inhibitor of protein synthesis, the rates of cell death and MnSOD degradation were accelerated. Fas-induced MnSOD cleavage was partially inhibited in the presence of the pan-caspase inhibitor, z-VAD-fmk. MnSOD in the mitochondrial fractions was cleaved in vitro by treatment with the cytosolic fraction of Fas-activated cells. Moreover, two possible cleavage sites of recombinant hMnSOD by direct interaction with recombinant caspase-3 were noted. Cellular and mitochondrial factors were found to be necessary for the interaction. These factors include intracellular mobilization of calcium. Our data indicate that inactivation of MnSOD in receptor-mediated apoptosis by caspase-specific degradation would render the mitochondria sensitive to the steady-state production of superoxide, decrease the steady-state flux of H(2)O(2), expedite the loss of mitochondrial function, and potentiate apoptosis.

Manganese superoxide dismutase inactivation during Fas (CD95)-mediated apoptosis in Jurkat T cells.

Selenium is recognized as essential in animal and human nutrition. Several hypotheses have been advanced for its biological activity. The aim of this study was to investigate the in vivo effect of selenium on rat liver manganese superoxide dismutase (MnSOD), a key antioxidant enzyme, under naïve and inflammatory conditions. Rats received sodium selenite supplementation and LPS injection. Whole-liver samples, isolated hepatocytes, Kupffer cells and blood samples were subjected to protein, RNA and biochemical analysis. Liver enrichment with selenium increased whole-liver MnSOD levels due to an increase in MnSOD transcription in hepatocytes. This was due to an increase in the ratio of specificity protein 1 to activating enhancer binding protein 2 DNA-binding activity. The inflammatory stimulus further elevated MnSOD levels in the whole-liver that was abrogated in sodium selenite supplementation due to reduced transcription of MnSOD in Kupffer cells. Moreover, selenium enrichment decreased Kupffer cells IL-6 transcription in LPS-injected animals. Anti-inflammatory activity of selenium was demonstrated by normalized blood levels of ALT and IL-6 in LPS-injected animals. In conclusion, selenium up-regulates hepatocytes MnSOD expression, probably improving their anti-oxidant defense, while decreasing MnSOD and IL-6 transcription in Kupffer cells in the presence of inflammatory stimuli, attenuating their inflammatory response. This selective mechanism may explain the anti-inflammatory and hepato-protective effect of selenium.

Selenium supplementation increases liver MnSOD expression: molecular mechanism for hepato-protection.

Hepatic ischemia-reperfusion (I/R) injury occurs in a variety of clinical settings and generates the release of endogenous noninfectious ligands called damage-associated molecular pattern (DAMP) signal molecules from damaged cells. This study investigates the effect of DAMP molecules released by Kupffer cells (KC) in I/R injury on the expression of liver manganese superoxide dismutase (MnSOD), a key mitochondrial antioxidant enzyme. We show that MnSOD expression levels are increased in rats and remain high for 24 h after 30 min of ischemia. KC were damaged and depleted after I/R, in association with MnSOD upregulation. Causality was established by treatment with gadolinium chloride, known to selectively destroy KC, which also increased MnSOD levels. Recovery from the early damage (6 h) to the liver tissue was evidenced after 24 h. A physiological protective role for MnSOD was also confirmed by the increased susceptibility of MnSOD-knockdown AML-12 hepatocyte cells to I/R-induced cell death. Inhibition of DAMP molecule high-mobility group box-1 activity by injection of neutralizing antibody partially abolished the increase in liver MnSOD after I/R. Direct injection of ATP, to animals or cells, stimulated MnSOD upregulation. Another DAMP molecule, monosodium urate, also induced MnSOD expression in hepatocyte AML-12 and FaO cell cultures. In conclusion, a connection between danger signals and upregulation of the antioxidant defense system is shown here for the first time in the context of I/R liver injury.

Antioxidant defense in hepatic ischemia-reperfusion injury is regulated by damage-associated molecular pattern signal molecules.

This study investigates the mechanism by which MnSOD exerts its protective effect in hypoxia-reoxygenation (H/R) injury in hepatocytes. Following induction of H/R, MnSOD expression and activity levels increased and remained high for over 24 h. Hepatocytes silenced for MnSOD (siMnSOD) demonstrated increased susceptibility to H/R-induced apoptotic cell death and a lower capacity to generate mitochondrial reactive oxygen species. Microarray and real time PCR analysis of gene expression from siMnSOD cells revealed a number of down-regulated protective genes, including hemeoxygenase-1, glutamate-cysteine ligase and Nrf2, a master regulator of cellular adaptation to stress. Decreased Nrf2 protein expression and nuclear translocation were also confirmed in siMnSOD cells. siMnSOD cells showed low glutathione (GSH) content with no oxidation to GSSG, lower lipid peroxidation levels than their controls and lower mitochondrial membrane potential, which all were even more salient after H/R. Therefore, MnSOD appears to act as a signalling mediator for the activation of survival genes following H/R injury in hepatocytes.

Protective signalling effect of manganese superoxide dismutase in hypoxia-reoxygenation of hepatocytes.

The cytotoxicity of tungsten disulfide nano tubes (INT-WS2) and inorganic fullerene-like molybdenum disulfide (IF-MoS2) nanoparticles (NPs) used in industrial and medical applications was evaluated in comparison to standard environmental particulate matter. The IF-MoS2 and INT-WS2 reside in vesicles/inclusion bodies, suggestive of endocytic vesicles. In cells representing the respiratory, immune and metabolic systems, both IF-MoS2 and INT-WS2 NPs remained nontoxic compared to equivalent concentrations (up to 100 μg/mL in the medium) of silica dioxide (SiO2), diesel engine-derived and carbon black NPs, which induced cell death. Associating with this biocompatibility of IF-MoS2\INT-WS2, we demonstrate in nontransformed human bronchial cells (NL-20) relative low induction of the pro-inflammatory cytokines IL-1β, IL-6, IL-8, and TNF-α. Moreover, IF-MoS2 and INT-WS2 activated antioxidant response as measured by the antioxidant response element (ARE) using a luciferase reporter, and induced Nrf2-mediated Phase II detoxification genes. Collectively, our findings suggest that the lower cytotoxicity of IF-MoS2 and INT-WS2 NPs does not reflect general biological inertness. Rather, compared to other NP's, it likely results from decreased pro-inflammatory activation, but a comparable significant capacity to induce protective antioxidant/detoxification defense mechanisms.

Low cytotoxicity of inorganic nanotubes and fullerene-like nanostructures in human bronchial epithelial cells: relation to inflammatory gene induction and antioxidant response.

Inhalation of traffic-associated atmospheric particulate matter (PM2.5) is recognized as a significant health risk. In this study, we focused on a single ("subclinical response") exposure to water-soluble extracts from PM collected at a roadside site in a major European city to elucidate potential components that drive pulmonary inflammatory, oxidative, and defense mechanisms and their systemic impacts. Intratracheal instillation (IT) of the aqueous extracts induced a 24 h inflammatory response characterized by increased broncho-alveolar lavage fluid (BALF) cells and cytokines (IL-6 and TNF-α), increased reactive oxygen species production, but insignificant lipids and proteins oxidation adducts in mouse lungs. This local response was largely self-resolved by 48 h, suggesting that it could represent a subclinical response to everyday-level exposure. Removal of soluble metals by chelation markedly diminished the pulmonary PM-mediated response. An artificial metal solution (MS) recapitulated the PM extract response. The self-resolving nature of the response is associated with activating defense mechanisms (increased levels of catalase and glutathione peroxidase expression), observed with both PM extract and MS. In conclusion, metals present in PM collected near roadways are largely responsible for the observed transient local pulmonary inflammation and oxidative stress. Simultaneous activation of the antioxidant defense response may protect against oxidative damage.

Single Exposure to near Roadway Particulate Matter Leads to Confined Inflammatory and Defense Responses: Possible Role of Metals.

Exposure to particulate matter (PM) pollution in cities and urban canyons can be harmful to the exposed population. However, the underlying mechanisms that lead to health effects are not yet elucidated. It is postulated that exposure to repeated, small, environmentally relevant concentrations can affect lung homeostasis. This study examines the impact of repeated exposures to urban PM on mouse lungs with focus on inflammatory and oxidative stress parameters. Aqueous extracts from collected urban PM were administered to mice by 5 repeated intra-tracheal instillations (IT). Multiple exposures, led to an increase in cytokine levels in both bronchoalveolar lavage fluid and in the blood serum, indicating a systemic reaction. Lung mRNA levels of antioxidant/phase II detoxifying enzymes decreased by exposure to the PM extract, but not when metals were removed by chelation. Finally, disruption of lung tissue oxidant-inflammatory/defense balance was evidenced by increased levels of lipid and protein oxidation. Unlike response to a single IT exposure to the same dose and source of extract, multiple exposures result in lung oxidative damage and a systemic inflammatory reaction. These could be attributed to compromised capacity to activate the protective Nrf2 tissue defense system. It is suggested that water-soluble metals present in urban PM, potentially from break and tire wear, may constitute major drivers of the pulmonary and systemic responses to multiple exposure to urban PM.

Repeated exposures to roadside particulate matter extracts suppresses pulmonary defense mechanisms, resulting in lipid and protein oxidative damage.

In this study we investigated the possible causal role for soluble metal species extracted from roadway traffic emissions in promoting particulate matter (PM)-induced reactive oxygen species (ROS) production and antioxidant response element (ARE) promoter activation. To this end, these responses have been evaluated in alveolar macrophage and epithelial lung cells that have been exposed to 'Unfiltered', 'Filtered' and 'Filtered+Chelexed' water extracts of PM samples collected from the roadway urban environments of Thessaloniki, Milan and London. Except for Thessaloniki, our results demonstrate that filtration resulted in a minor decrease in ROS activity of the fine PM fraction, suggesting that ROS activity is attributed mainly to water-soluble PM species. In contrast to ROS, ARE activity was mediated predominantly by the water-soluble component of PM present in both the fine and coarse extracts. Further removal of metals by Chelex treatment from filtered water extracts showed that soluble metal species are the major factors mediating ROS and ARE activities of the soluble fraction, especially in the London PM extracts. Finally, utilizing step-wise multiple-regression analysis, we show that 87% and 78% of the total variance observed in ROS and ARE assays, respectively, is accounted for by changes in soluble metal concentration. Using a statistical analysis we find that As, Zn and Fe best predict the ROS-generating/ARE-activating capacity of the near roadway particulate matter in the pulmonary cells studied. Collectively, our findings imply that soluble metals present in roadside PM are potential drivers of both pro- and anti-oxidative effects of PM in respiratory tract.

Article	Year
AlphaMUPA mice: a transgenic model for longevity induced by caloric restriction. Mechanisms of ageing and development\| PubMed ID: 15621205	2005
Long-lived alphaMUPA transgenic mice show reduced SOD2 expression, enhanced apoptosis and reduced susceptibility to the carcinogen dimethylhydrazine. Mechanisms of ageing and development\| PubMed ID: 16139868	2005
Manganese superoxide dismutase inactivation during Fas (CD95)-mediated apoptosis in Jurkat T cells. Free radical biology & medicine\| PubMed ID: 17157182	2006
Selenium supplementation increases liver MnSOD expression: molecular mechanism for hepato-protection. Journal of inorganic biochemistry\| PubMed ID: 17804075	2008
Antioxidant defense in hepatic ischemia-reperfusion injury is regulated by damage-associated molecular pattern signal molecules. Free radical biology & medicine\| PubMed ID: 18675899	2008
Protective signalling effect of manganese superoxide dismutase in hypoxia-reoxygenation of hepatocytes. Free radical research\| PubMed ID: 19905985	2009
Low cytotoxicity of inorganic nanotubes and fullerene-like nanostructures in human bronchial epithelial cells: relation to inflammatory gene induction and antioxidant response. Environmental science & technology\| PubMed ID: 24533583	2014
Single Exposure to near Roadway Particulate Matter Leads to Confined Inflammatory and Defense Responses: Possible Role of Metals. Environmental science & technology\| PubMed ID: 26121492	2015
Repeated exposures to roadside particulate matter extracts suppresses pulmonary defense mechanisms, resulting in lipid and protein oxidative damage. Environmental pollution (Barking, Essex : 1987)\| PubMed ID: 26735168	2015
ROS-generating/ARE-activating capacity of metals in roadway particulate matter deposited in urban environment. Environmental research\| PubMed ID: 26775006	2016

Michal Pardo

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