A Macrophage-Based Method for the Assessment of the Reactive Oxygen Species (ROS) Activity of Atmospheric Particulate Matter (PM) and Application to Routine (Daily-24 h) Aerosol Monitoring Studies

Both short- and long-term exposure to particulate matter (PM) air pollution have been demonstrated to cause increases in cardiovascular disease, cancer, and respiratory disorders. Although the specific mechanisms by which exposure to PM cause these affects are unclear, significant evidence has accumulated to suggest that PM exposure leads to increased inflammation as the result of excessive production of reactive oxygen species (ROS) in critical cell types. In order to better understand how real-world PM exposure causes adverse health effects, there is a need to efficiently integrate metrics of PM toxicity into large scale air monitoring and health effects/epidemiology studies. Here we describe a rapid, inexpensive, method that can be employed to assess the potential of sub-mg masses of PM to generate oxidative stress in alveolar macrophage cells. Importantly, the approach is compatible with routine daily PM sampling programs such as those administered by EPA (Speciation trends network (STN), IMPROVE network, PM2.5 mass monitoring network), allowing for multiple samples to be assessed simultaneously with low volumes and brief exposure periods. We apply the method to a set of water extracts of daily PM2.5 samples (25–350 μ g PM mass) collected in the Denver-Metro area. Variations in the magnitude of the ROS response observed between the samples were only partially explained by differences in mass loading, with the highest levels of ROS being observed in samples collected during the summer months. This assay provides a very useful tool that can be coupled with detailed chemical analysis and statistical models to work towards the goal of attributing PM toxicity to specific real-world chemical sources.     

Vitamin C and Vitamin E in Prevention of Nonalcoholic Fatty Liver Disease (NAFLD) in Choline Deficient Diet Fed Rats

Aim  

Oxidative stress has been implicated in the pathogenesis of Nonalcoholic Fatty Liver Disease (NAFLD). Vitamin C and vitamin E are known to react with reactive oxygen species (ROS) blocking the propagation of radical reactions in a wide range of oxidative stress situations. The potential therapeutic efficacy of antioxidants in NAFLD is unknown. The aim of this study was to evaluate the role of antioxidant drugs (vitamin C or vitamin E) in its prevention.     

Role of pyrite in formation of hydroxyl radicals in coal: possible implications for human health

Background  

The harmful effects from inhalation of coal dust are well-documented. The prevalence of lung disease varies by mining region and may, in part, be related to regional differences in the bioavailable iron content of the coal. Pyrite (FeS₂), a common inorganic component in coal, has been shown to spontaneously form reactive oxygen species (ROS) (i.e., hydrogen peroxide and hydroxyl radicals) and degrade nucleic acids. This raises the question regarding the potential for similar reactivity from coal that contains pyrite. Experiments were performed to specifically evaluate the role of pyrite in coal dust reactivity. Coal samples containing various amounts of FeS₂ were compared for differences in their generation of ROS and degradation of RNA.     

In vitro effects of nanoparticles on renal cells

Background  

The ability of nanoparticles to cross the lung-blood barrier suggests that they may translocate to blood and to targets distant from their portal of entry. Nevertheless, nanotoxicity in organs has received little attention. The purpose of this study was to evaluate nanotoxicity in renal cells using in vitro models. Various carbon black (CB) (FW2–13 nm, Printex60-21 nm and LB101-95 nm) and titanium dioxide (TiO₂-15 and TiO₂-50 nm) nanoparticles were characterized on size by electron microscopy. We evaluated theirs effects on glomerular mesangial (IP15) and epithelial proximal tubular (LLC-PK₁) renal cells, using light microscopy, WST-1 assay, immunofluorescence labeling and DCFH-DA for reactive oxygen species (ROS) assay.     

ROS-mediated TNF-α and MIP-2 gene expression in alveolar macrophages exposed to pine dust

Background  

Respiratory symptoms, impaired lung function, and asthma have been reported in workers exposed to wood dust in a number of epidemiological studies. The underlying pathomechanisms, however, are not well understood. Here, we studied the effects of dust from pine (PD) and heat-treated pine (HPD) on the release of reactive oxygen species (ROS) and inflammatory mediators in rat alveolar macrophages.     

Comparison of the Chemical and Oxidative Characteristics of Particulate Matter (PM) Collected by Different Methods: Filters,Impactors, and BioSamplers

In this study, we compare the chemical and oxidative characteristics of concentration-enriched PM_2.5 samples simultaneously collected by a filter, a Nano-Micro-Orifice Uniform Deposition Impactor, and a BioSampler. Gravimetric measurements showed considerable agreement in particulate matter (PM) collection efficiency for all three samplers. Accordingly, samples from the three collectors exhibited similar chemical compositions. The mass fractions of their inorganic ions, labile and nonlabile, were comparable. Moreover, the organic carbon (OC) content of the BioSampler slurry was similar to that of the filter, while water-soluble OC levels of the filter and impactor samples were close to a 100% agreement. Lastly, linear regression analyses demonstrated that the water-soluble elements existed in similar proportions for the filter and impactor samples. Their respective total components were also in very good agreement. By contrast, the recoverable elements from the BioSampler slurry, determined by high-resolution magnetic sector inductively coupled plasma mass spectrometry, were in good agreement with the water-soluble elements of the filter and impactor samples but not their corresponding total components. In spite of the overall agreement among the samples on their chemical composition, findings from a macrophage reactive oxygen species (ROS), a dithiothreitol (DTT), and a dihydroxybenzoate (DHBA) assay revealed that the oxidative potential of aqueous extracts of the filter and impactor substrates was similar yet substantially lower than that of the BioSampler slurry. However, filtering of the BioSampler slurry, i.e., removal of insoluble PM components, attenuated its ROS activity to about the same level as that of the water extracts of the filter and impactor samples. These findings first indicate that insoluble PM species are potentially redox active, and second that particle collection by the BioSampler, which circumvents the need for PM extraction, constitutes a viable alternative for collecting concentrated particles for characterization of the oxidative properties of PM.     

Cocoa intake and arterial stiffness in subjects with cardiovascular risk factors

Background

DNA is constantly exposed to reactive oxygen species (ROS), spontaneously arising during the normal oxygen metabolism. ROS may result in temporary as well as permanent modifications in various cellular components such as lipids, proteins and DNA, which may have deleterious consequences. Demonstrating that a dietary supplementation of antioxidants can reduce oxidative DNA damage may provide evidence for the value of such supplementation in prevention of cancer and age related diseases.

Findings

The present study was conducted to address whether tomato juice protects against ROS induced by extensive physical exercise in untrained individuals. As a marker of oxidative stress, serum levels of 8-oxodG were monitored using a modified ELISA. An intervention was performed involving 15 untrained healthy subjects who performed a 20?min physical exercise at 80% of maximum pulse using an ergometer bicycle. Blood samples were taken before and one hour after the exercise. The procedure was repeated after 5?weeks with a daily intake of 150?ml tomato juice and followed by a 5?weeks wash-out period and another 5?weeks with a daily intake of tomato juice. The results indicated that a daily intake of tomato juice, equal to 15?mg lycopene per day, for 5?weeks significantly reduced the serum levels of 8-oxodG after an extensive physical exercise.

Conclusion

These data strongly suggest that tomato juice has a potential antioxidant effect and may reduce the elevated level of ROS induced by oxidative stress.     

Shape matters: effects of silver nanospheres and wires on human alveolar epithelial cells

Background

Ambient particulate matter (PM) exposure is associated with respiratory and cardiovascular morbidity and mortality. To what extent such effects are different for PM obtained from different sources or locations is still unclear. This study investigated the in vitro toxicity of ambient PM collected at different sites in the Netherlands in relation to PM composition and oxidative potential.

Method

PM was sampled at eight sites: three traffic sites, an underground train station, as well as a harbor, farm, steelworks, and urban background location. Coarse (2.5-10 μm), fine (< 2.5 μm) and quasi ultrafine PM (qUF; < 0.18 μm) were sampled at each site. Murine macrophages (RAW 264.7 cells) were exposed to increasing concentrations of PM from these sites (6.25-12.5-25-50-100 μg/ml; corresponding to 3.68-58.8 μg/cm²). Following overnight incubation, MTT-reduction activity (a measure of metabolic activity) and the release of pro-inflammatory markers (Tumor Necrosis Factor-alpha, TNF-α; Interleukin-6, IL-6; Macrophage Inflammatory Protein-2, MIP-2) were measured. The oxidative potential and the endotoxin content of each PM sample were determined in a DTT- and LAL-assay respectively. Multiple linear regression was used to assess the relationship between the cellular responses and PM characteristics: concentration, site, size fraction, oxidative potential and endotoxin content.

Results

Most PM samples induced a concentration-dependent decrease in MTT-reduction activity and an increase in pro-inflammatory markers with the exception of the urban background and stop & go traffic samples. Fine and qUF samples of traffic locations, characterized by a high concentration of elemental and organic carbon, induced the highest pro-inflammatory activity. The pro-inflammatory response to coarse samples was associated with the endotoxin level, which was found to increase dramatically during a three-day sample concentration procedure in the laboratory. The underground samples, characterized by a high content of transition metals, showed the largest decrease in MTT-reduction activity. PM size fraction was not related to MTT-reduction activity, whereas there was a statistically significant difference in pro-inflammatory activity between Fine and qUF PM. Furthermore, there was a statistically significant negative association between PM oxidative potential and MTT-reduction activity.

Conclusion

The response of RAW264.7 cells to ambient PM was markedly different using samples collected at various sites in the Netherlands that differed in their local PM emission sources. Our results are in support of other investigations showing that the chemical composition as well as oxidative potential are determinants of PM induced toxicity in vitro.     

Development and Evaluation of a High-Volume Aerosol-into-Liquid Collector for Fine and Ultrafine Particulate Matter

This study presents a novel high-volume aerosol-into-liquid collector, developed to provide concentrated slurries of fine and/or ultrafine particulate matter (PM) to be used for unattended, in situ measurements of PM chemistry and toxicity. This system operates at 200 liters per minute (L/min) flow and utilizes the saturation–condensation, particle-to-droplet growth component of the versatile aerosol concentration enrichment system (VACES), growing fine or ultrafine PM to 3–4-μm droplets, in conjunction with a newly designed impactor, in which grown particles are collected gradually forming highly concentrated slurries. Laboratory evaluation results indicated an excellent overall system collection efficiency (over 90%) for both monodisperse and polydisperse particles in the range of 0.01 to 2 μm. Field evaluations illustrated that overall a very good agreement was obtained for most PM_2.5 species between the new aerosol collection system and the VACES/BioSampler tandem as well as filter samplers operating in parallel. Very good agreement between the new system and the VACES/BioSampler was also observed for reactive oxygen species (ROS) in ambient PM_2.5 samples, whereas lower ROS values were obtained from the water extracts of the filter, likely due to incomplete extraction of water insoluble redox active species collected on the filter substrate. Moreover, the field tests indicated that the new aerosol collection system could achieve continuous and unattended collection of concentrated suspensions for at least 2 to 3 days without any obvious shortcomings in its operation. Both laboratory and field evaluations of the high-volume aerosol-into-liquid collector suggest that this system is an effective technology for collection and characterization of ambient aerosols.

Copyright 2013 American Association for Aerosol Research     

The Effect of Coating TiO<Subscript>2</Subscript> on the CO Oxidation of the Pt/γ-Alumina Catalysts

Abstract  

The effect of coating TiO₂ on the CO oxidation of the Pt/γ-alumina catalysts was observed through activity tests and surface characterization spectroscopy by X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR) experiments. XPS results evidenced the occurrence of different Pt²⁺ species and metallic Pt⁰ at the surface which suggest electron transfer of titanium (cation) to the platinum atom and the reduction of titanium (Ti⁴⁺ → Ti³⁺). FTIR analyses suggested oxygen spillover mechanism at the interface between titanium dioxide and platinum that may explain the catalytic activity of the platinum titania-supported catalysts. The apparent activation energy for the CO oxidation was 52.5 kJ/mol and similar for all catalysts. However, the frequency factor changed significantly, indicating interfacial phenomena caused by CO and oxygen adsorptions over TiO _x species and Al₂O₃ support with similar dispersions.     

Electron transfer pathways in microbial oxygen biocathodes

The ability of some bacteria to enhance the rate of cathodic oxygen reduction to water has been recently discovered, opening the way to an entirely renewable and environmentally friendly concept of biocathode. In this study we reveal that several mechanisms may induce catalytic effects by bacteria. These comprise mechanisms that are putatively beneficial to the bacteria as well as mechanisms which are merely side effects, including quinone autoxidation and direct O₂ reduction by heme compounds. Here we showed that 1 μM of ACNQ is able to generate a significant catalytic wave for oxygen reduction, with onset at approximately 0 V vs. SHE. Similarly, adsorption of hemin on a carbon surface catalyses O₂ reduction to H₂O₂ with an onset of +0.2 V vs. SHE. To evaluate the catalytic pathways of live cells on cathodic oxygen reduction, two species of electrochemically active bacteria were selected as pure cultures, namely Acinetobacter calcoaceticus and Shewanella putrefaciens. The former appears to exploit a self-excreted redox compound with redox characteristics matching those of pyrroloquinoline quinone (PQQ) for extracellular electron transfer. The latter appears to utilise outer membrane-bound redox compounds. Interaction of quinones and cytochromes with the membrane-bound electron transfer chain is yet to be proven.     

Differential effects of diesel exhaust particles on T cell differentiation and autoimmune disease

Background

Exposure to particulate matter (PM) has been associated with increased incidence and severity of autoimmune disease. Diesel PM is primarily composed of an elemental carbon core and adsorbed organic compounds such as polycyclic aromatic hydrocarbons (PAHs) and contributes up to 40% of atmospheric PM. The organic fraction (OF) of PM excludes all metals and inorganics and retains most organic compounds, such as PAHs. Both PM and OF increase inflammation in vitro and aggravate autoimmune disease in humans. PAHs are known aryl hydrocarbon receptor (AHR) ligands. The AHR modulates T cell differentiation and effector function in vitro and in experimental autoimmune encephalomyelitis (EAE), a murine model of autoimmune disease. This study aims to identify whether the total mass or active components of PM are responsible for activating pathways associated with exposure to PM and autoimmune disease. This study tests the hypothesis that active components present in diesel PM and their OF enhance effector T cell differentiation and aggravate autoimmune disease.

Results

Two different diesel samples, each characterized for their components, were tested for their effects on autoimmunity. Both diesel PM enhanced effector T cell differentiation in an AHR-dose-dependent manner and suppressed regulatory T cell differentiation in vitro. Both diesel PM aggravated EAE in vivo. Fractionated diesel OFs exhibited the same effects as PM in vitro, but unlike PM, only one diesel OF aggravated EAE. Additionally, both synthetic PAH mixtures that represent specific PAHs found in the two diesel PM samples enhanced Th17 differentiation, however one lost this effect after metabolism and only one required the AHR.

Conclusions

These findings suggest that active components of PM and not total mass are driving T cell responses in vitro, but in vivo the PM matrix and complex mixtures adsorbed to the particles, not just the OF, are contributing to the observed EAE effects. This implies that examining OF alone may not be sufficient in vivo. These data further suggest that bioavailability and metabolism of organics, especially PAHs, may have an important role in vivo.

     

Sterols from Mytilidae Show Anti-Aging and Neuroprotective Effects via Anti-Oxidative Activity

For screening anti-aging samples from marine natural products, K6001 yeast strain was employed as a bioassay system. The active mussel extract was separated to give an active sterol fraction (SF). SF was further purified, and four sterol compounds were obtained. Their structures were determined to be cholesterol (CHOL), brassicasterol, crinosterol, and 24-methylenecholesterol. All compounds showed similar anti-aging activity. To understand the action mechanism involved, anti-oxidative experiments, reactive oxygen species (ROS) assays, and malondialdehyde (MDA) tests were performed on the most abundant compound, CHOL. Results indicated that treatment with CHOL increases the survival rate of yeast under oxidative stress and decreases ROS and MDA levels. In addition, mutations of uth1, skn7, sod1, and sod2, which feature a K6001 background, were employed and the lifespans of the mutations were not affected by CHOL. These results demonstrate that CHOL exerts anti-aging effects via anti-oxidative stress. Based on the connection between neuroprotection and anti-aging, neuroprotective experiments were performed in PC12 cells. Paraquat was used to induce oxidative stress and the results showed that the CHOL and SF protect the PC12 cells from the injury induced by paraquat. In addition, these substance exhibited nerve growth factor (NGF) mimic activities again confirmed their neuroprotective function.     

Reactive oxygen species generation and dispersant-dependent electron transfer through single-walled carbon nanotubes in water

Although induction of oxidative stress is widely accepted as one of the major cytotoxic effects of carbon nanotubes (CNTs), there is no solid understanding of how biological redox reactions are affected and how reactive oxygen species (ROS) are generated by CNTs, especially when they are coated with various dispersing agents. In this study, we investigated electron transfer from biological reducing agents through nonfunctionalized single-walled carbon nanotubes (SWCNTs) to molecular oxygen, generating ROS in the process. Electron transfer rates in the colloidal SWCNT suspensions depended on the dispersant used to stabilize them, with six dispersants examined. Oxidation of both nicotinamide adenine dinucleotide (NADH) and dithiothreitol was catalyzed by SWCNTs coated with either cetyltrimethylammonium bromide (CTAB) or Suwannee River natural organic matter (SRNOM). SWCNTs coated with other types of surfactants showed only slight effect. In the presence of NADH or dithiothreitol, generation of ROS also was dispersant-dependent, with CTAB- and SRNOM-coated SWCNTs generating significant amounts of superoxide anion and hydrogen peroxide. In systems containing xanthine and xanthine oxidase, accumulated charge on the SWCNTs appeared to be transferred to superoxide anion, resulting in indirect disproportionation of superoxide anion, forming more hydrogen peroxide.     

Germinated Rhynchosia nulubilis Fermented with Lactobacillus pentosus SC65 Reduces Particulate Matter Induced Type II Alveolar Epithelial Apoptotic Cell Death

Particulate matter (PM) is a significant environmental pollutant that promotes respiratory diseases, including lung injury and inflammation, by inducing oxidative stress. Rhynchosia nulubilis (black soybean) is traditionally used to prevent chronic respiratory disease via inducing antioxidant and anti-inflammatory effects. To investigate the effects of Lactobacillus pentosus SC65 fermented GR (GR-SC65) and Pediococcus pentosaceus ON81A (GR-ON81A) against PM-induced oxidative stress and cell death in A549 cells, we performed the 2-7-dichlorodihydrofluorescein diacetate and cell counting kit-8 assays, as well as Hoechst 33342 and propidium iodide staining and western blotting. GR-SC65 showed the highest total polyphenolic contents and 1,1-diphenyl-2-picrylidrazil radical scavenging activity among lactic acid bacteria-fermented GRs (p < 0.001 vs. GR). Four soy peptides, β-conglycinin breakdowns (INAENNQRNF, ISSEDKPFN, LAFPGSAQAVEK, and LAFPGSAKDIEN), were detected in GR-SC65, but not in GR. In GR-SC65, PM-induced A549 cell death was less than that observed in GR-ON81A and GR (p < 0.001 vs. PM-treated group). GR-SC65 significantly decreased intracellular reactive oxidative species (ROS) when compared with PM (*** p < 0.001 vs. PM). GR-SC65 decreased the levels of BAX, active caspase-9, -3, and poly ADP-ribose polymerase (PARP) proteins (^# p < 0.01, ^### p < 0.001 vs. PM), while increasing the level of BCL-2 protein, a mitochondrial anti-apoptotic protein (^### p < 0.001 vs. PM). Our findings indicate that GR-SC65 inhibited PM-induced cell death by suppressing the levels of ROS, active caspase-9 and -3, and PARP proteins, while enhancing the level of BCL-2 protein in type II alveolar epithelial A549 cells. Therefore, GR-SC65 might be a potential therapeutic and preventive agent against PM-induced lung injury.     

Implication of scavenger receptors in the interactions between diesel exhaust particles and immature or mature dendritic cells

Background

Engineered iron nanoparticles are being explored for the development of biomedical applications and many other industry purposes. However, to date little is known concerning the precise mechanisms of translocation of iron nanoparticles into targeted tissues and organs from blood circulation, as well as the underlying implications of potential harmful health effects in human.

Results

The confocal microscopy imaging analysis demonstrates that exposure to engineered iron nanoparticles induces an increase in cell permeability in human microvascular endothelial cells. Our studies further reveal iron nanoparticles enhance the permeability through the production of reactive oxygen species (ROS) and the stabilization of microtubules. We also showed Akt/GSK-3β signaling pathways are involved in iron nanoparticle-induced cell permeability. The inhibition of ROS demonstrate ROS play a major role in regulating Akt/GSK-3β – mediated cell permeability upon iron nanoparticle exposure. These results provide new insights into the bioreactivity of engineered iron nanoparticles which can inform potential applications in medical imaging or drug delivery.

Conclusion

Our results indicate that exposure to iron nanoparticles induces an increase in endothelial cell permeability through ROS oxidative stress-modulated microtubule remodeling. The findings from this study provide new understandings on the effects of nanoparticles on vascular transport of macromolecules and drugs.     

Facile Oxidation of Leucomethylene Blue and Dihydroflavins by Artemisinins: Relationship with Flavoenzyme Function and Antimalarial Mechanism of Action

The antimalarial drug methylene blue (MB) affects the redox behaviour of parasite flavin‐dependent disulfide reductases such as glutathione reductase (GR) that control oxidative stress in the malaria parasite. The reduced flavin adenine dinucleotide cofactor FADH₂ initiates reduction to leucomethylene blue (LMB), which is oxidised by oxygen to generate reactive oxygen species (ROS) and MB. MB then acts as a subversive substrate for NADPH normally required to regenerate FADH₂ for enzyme function. The synergism between MB and the peroxidic antimalarial artemisinin derivative artesunate suggests that artemisinins have a complementary mode of action. We find that artemisinins are transformed by LMB generated from MB and ascorbic acid (AA) or N‐benzyldihydronicotinamide (BNAH) in situ in aqueous buffer at physiological pH into single electron transfer (SET) rearrangement products or two‐electron reduction products, the latter of which dominates with BNAH. Neither AA nor BNAH alone affects the artemisinins. The AA–MB SET reactions are enhanced under aerobic conditions, and the major products obtained here are structurally closely related to one such product already reported to form in an intracellular medium. A ketyl arising via SET with the artemisinin is invoked to explain their formation. Dihydroflavins generated from riboflavin (RF) and FAD by pretreatment with sodium dithionite are rapidly oxidised by artemisinin to the parent flavins. When catalytic amounts of RF, FAD, and other flavins are reduced in situ by excess BNAH or NAD(P)H in the presence of the artemisinins in the aqueous buffer, they are rapidly oxidised to the parent flavins with concomitant formation of two‐electron reduction products from the artemisinins; regeneration of the reduced flavin by excess reductant maintains a catalytic cycle until the artemisinin is consumed. In preliminary experiments, we show that NADPH consumption in yeast GR with redox behaviour similar to that of parasite GR is enhanced by artemisinins, especially under aerobic conditions. Recombinant human GR is not affected. Artemisinins thus may act as antimalarial drugs by perturbing the redox balance within the malaria parasite, both by oxidising FADH₂ in parasite GR or other parasite flavoenzymes, and by initiating autoxidation of the dihydroflavin by oxygen with generation of ROS. Reduction of the artemisinin is proposed to occur via hydride transfer from LMB or the dihydroflavin to O1 of the peroxide. This hitherto unrecorded reactivity profile conforms with known structure–activity relationships of artemisinins, is consistent with their known ability to generate ROS in vivo, and explains the synergism between artemisinins and redox‐active antimalarial drugs such as MB and doxorubicin. As the artemisinins appear to be relatively inert towards human GR, a putative model that accounts for the selective potency of artemisinins towards the malaria parasite also becomes apparent. Decisively, ferrous iron or carbon‐centered free radicals cannot be involved, and the reactivity described herein reconciles disparate observations that are incompatible with the ferrous iron–carbon radical hypothesis for antimalarial mechanism of action. Finally, the urgent enquiry into the emerging resistance of the malaria parasite to artemisinins may now in one part address the possibilities either of structural changes taking place in parasite flavoenzymes that render the flavin cofactor less accessible to artemisinins or of an enhancement in the ability to use intra‐erythrocytic human disulfide reductases required for maintenance of parasite redox balance.     

Catalytic Reactions of Guaiacol: Reaction Network and Evidence of Oxygen Removal in Reactions with Hydrogen

Abstract  

The conversion of guaiacol, a prototypical compound representative of lignin-derived pyrolysis bio-oils, was catalyzed by Pt/Al₂O₃ in the presence of H₂ at 573 K. The conversion took place with a high selectivity for aromatic carbon–oxygen bond cleavage relative to the accompanying methyl group transfer reactions. This oxygen removal was not observed in the absence of H₂ as a co-reactant. Products that were formed by methyl-group transfer match those produced in the conversion catalyzed by zeolite HY, which was not active for oxygen removal reactions.     

Tiron Has Negative Effects on Osteogenic Differentiation via Mitochondrial Dysfunction in Human Periosteum-Derived Cells

Tiron is a potent antioxidant that counters the pathological effects of reactive oxygen species (ROS) production due to oxidative stress in various cell types. We examined the effects of tiron on mitochondrial function and osteoblastic differentiation in human periosteum-derived cells (hPDCs). Tiron increased mitochondrial activity and decreased senescence-associated β-galactosidase activity in hPDCs; however, it had a detrimental effect on osteoblastic differentiation by reducing alkaline phosphatase (ALP) activity and alizarin red-positive mineralization, regardless of H₂O₂ treatment. Osteoblast-differentiating hPDCs displayed increased ROS production compared with non-differentiating hPDCs, and treatment with tiron reduced ROS production in the differentiating cells. Antioxidants decreased the rates of oxygen consumption and ATP production, which are increased in hPDCs during osteoblastic differentiation. In addition, treatment with tiron reduced the levels of most mitochondrial proteins, which are increased in hPDCs during culture in osteogenic induction medium. These results suggest that tiron exerts negative effects on the osteoblastic differentiation of hPDCs by causing mitochondrial dysfunction.