Search Results (49)

This study examines long-term trends in fine particulate matter (PM_2.5) composition and oxidative potential in Los Angeles based on data from the University of Southern California’s Particle Instrumentation Unit, with chemical composition retrieved from the EPA’s Air Quality System (AQS). While regulatory interventions have reduced PM_2.5 mass concentration and primary combustion-related components, our findings reveal a more complex toxicity pattern. From 2001 to 2008, the PM_2.5 oxidative potential, measured via the dithiothreitol (DTT) assay, declined from ~0.84 to ~0.16 nmol/min/m³ under stringent tailpipe controls. However, after this initial decline, PM_2.5 DTT stabilized and gradually increased from ~0.35 in 2012 to ~0.97 nmol/min/m³ by 2024, reflecting the growing influence of non-tailpipe emissions such as brake/tire wear. Metals, such as iron (Fe, ~150 ng/m³) and zinc (Zn, ~10 ng/m³), remained relatively stable as organic and elemental carbon (OC and EC) declined, resulting in non-tailpipe contributions dominating PM_2.5 toxicity. Although PM_2.5 mass concentrations were effectively reduced, the growing contribution of non-tailpipe emissions (e.g., brake/tire wear and secondary organic aerosols) underscores the limitations of mass-based standards and tailpipe-focused strategies. Our findings emphasize the need to broaden regulatory strategies, targeting emerging sources that shape PM_2.5 composition and toxicity and ensuring more improvements in public health outcomes. Full article

Environmental particulate matter (PM) exposure has been widely recognized for its significant adverse effects on human health. Monitoring PM levels is one of the essential parameters of air quality assessment. However, PM mass concentration alone does not sufficiently explain its toxicological impacts and effects on health. This study highlights the importance of oxidative potential (OP) as a promising metric for evaluating PM toxicity. It focuses on standardizing the dithiothreitol (DTT) assay as a tool for OP measurement. In order to investigate the impact of various extraction techniques, reagent concentrations, and assay conditions, four previously established protocols were tested without modification, while a novel protocol was introduced based on an extensive literature review. Results revealed strong positive correlations between the new and most established protocols. These findings highlight the significance of the new protocol in advancing the development of standardized methodologies for applying the DTT assay and demonstrating its reliability and relevance. While developing a standardized DTT assay involves addressing numerous parameters—from filter extraction to assay application—this research provides a solid base for achieving consistency in OP measurements and overcoming this critical issue. Full article

Cystathionine γ-lyase (CSE) is the second enzyme in the trans-sulfuration pathway that converts cystathionine to cysteine. It is also one of three major enzymes responsible for the biosynthesis of hydrogen sulfide (H₂S). CSE is believed to be the major source of endogenous H₂S in the cardiovascular system, and the CSE/H₂S system plays a crucial role in a variety of physiological and pathological processes. However, the regulatory mechanism of the CSE/H₂S system is less well understood, especially at the post-translational level. Here, we demonstrated that the persulfidation of CSE inhibits its activity by ~2-fold in vitro. The loss of this post-translational modification in the presence of dithiothreitol (DTT) results in a reversal of basal activity. Cys137 was identified as the site for persulfidation by combining mass spectrometry, mutagenesis, activity analysis and streptavidin–biotin pull-down assays. To test the physiological relevance of the persulfidation regulation of CSE, human aortic vascular smooth muscle cells (HA-VSMCs) were incubated with vascular endothelial growth factor (VEGF), which is known to enhance endogenous H₂S levels. Under these conditions, consistent with the change tendency of the cellular H₂S level, the CSE persulfidation levels increased transiently and then gradually decreased to the basal level. Collectively, our study revealed a negative feedback regulation mechanism of the CSE/H₂S system via the persulfidation of CSE and demonstrated the potential for maintaining cellular H₂S homeostasis under oxidative stress conditions, particularly in tissues where CSE is a major source of H₂S. Full article

Recent studies indicate that mass concentration alone is not the best parameter to assess the toxicity and the health effects of particulate matter (PM). Indeed, the chemical composition of the particles plays an important role, and oxidative potential (OP) measurements are being proposed as an alternative way to assess toxicity. The European Union (EU) is currently proposing a draft of the new air quality directive which includes OP measurements but does not specify the methods and/or protocols of analysis. In this light, the purpose of this study was to evaluate the feasibility of two literature assays, namely ascorbic acid (AA) and dithiothreitol (DTT), for routine PM analysis by testing urban PM filters from a one-year sampling campaign conducted by ARPA Lombardia. Indoor PM samples were also tested to emphasize the importance of monitoring closed spaces in which people spend most of their time. Following the optimization of the DTT assay, both methods proved to be suitable for large-scale PM analysis. The results show that the oxidative strength of urban PM is constant throughout the year for the outdoor samples (OP^AA: 0.067–0.39 nmol min⁻¹ m⁻³; OP^DTT: 0.033–0.109 nmol min⁻¹ m⁻³), indicating the need for routine OP monitoring. Instead, indoor areas were characterized by particles with a lower oxidative capacity (OP^AA: 5.40–24 pmol min⁻¹ m⁻³; OP^DTT: 9.7–32 pmol min⁻¹ m⁻³), driven both by lower concentrations and a different chemical composition. All the data collected highlight the need to add this parameter as part of the chemical characterization of PM, moving in the same direction as the new EU air quality directive. Full article

Pediatric neuroblastomas (NBs) are heterogeneous, aggressive, therapy-resistant embryonal tumors that originate from cells of neural crest origin committed to the sympathoadrenal progenitor cell lineage. Stress- and drug-resistance mechanisms drive post-therapeutic relapse and metastatic progression, the characterization and inhibition of which are major goals in improving therapeutic responses. Stress- and drug-resistance mechanisms in NBs include alternative TrkAIII splicing of the neurotrophin receptor tropomyosin-related kinase A (NTRK1/TrkA), which correlates with post-therapeutic relapse and advanced-stage metastatic disease. The TrkAIII receptor variant exerts oncogenic activity in NB models by mechanisms that include stress-induced mitochondrial importation and activation. In this study, we characterize novel targetable and non-targetable participants in this pro-survival mechanism in TrkAIII-expressing SH-SY5Y NB cells, using dithiothreitol (DTT) as an activator and a variety of inhibitors by regular and immunoprecipitation Western blotting of purified mitochondria and IncuCyte cytotoxicity assays. We report that stress-induced TrkAIII misfolding initiates this mechanism, resulting in Grp78, Ca²⁺-calmodulin, adenosine ribosylating factor (Arf) and Hsp90-regulated mitochondrial importation. TrkAIII imported into inner mitochondrial membranes is cleaved by Omi/high temperature requirement protein A2 (HtrA2) then activated by a mechanism dependent upon calmodulin kinase II (CaMKII), alpha serine/threonine kinase (Akt), mitochondrial Ca²⁺ uniporter and reactive oxygen species (ROS), involving inhibitory mitochondrial protein tyrosine phosphatase (PTPase) oxidation, resulting in phosphoinositide 3 kinase (PI3K) activation of mitochondrial Akt, which enhances stress resistance. This novel pro-survival function for misfolded TrkAIII mitigates the cytotoxicity of mitochondrial Ca²⁺ homeostasis disrupted during integrated stress responses, and is prevented by clinically approved Trk and Akt inhibitors and also by inhibitors of 78kDa glucose regulated protein (Grp78), heat shock protein 90 (Hsp90), Ca²⁺-calmodulin and PI3K. This identifies Grp78, Ca²⁺-calmodulin, Hsp90, PI3K and Akt as novel targetable participants in this mechanism, in addition to TrkAIII, the inhibition of which has the potential to enhance the stress-induced elimination of TrkAIII-expressing NB cells, with the potential to improve therapeutic outcomes in NBs that exhibit TrkAIII expression and activation. Full article

Oxidation potential (OP), reflecting the redox activities of particle matter (PM), is considered an optimal measure to explain the biological effects of PM exposure. However, the size resolution of the relationship between OP and chemical composition in PM, especially how the relationship changes after respiratory exposure, has not been well investigated. In this study, size-resolved indoor PM₁₀ samples were collected from a waste recycling plant from November to December 2021 using an Anderson eight-stage cascade impactor. OP, measured by a dithiothreitol (DTT) assay (defined as OP^DTT), and elements, determined by inductively coupled plasma–mass spectrometry (ICP-MS) in size-resolved PM, were determined to check their relationships and the related human exposure risk. The results indicated that compared with PM_0.4 and PM_0.4–2.1, PM_2.1–10 contributed the most to total OP^DTT and its bound elements contributed the most to potential health risks, both before and after respiratory exposure. The association between OP^DTT and the elements varied with PM size. Pearson correlation analysis showed that the PM_0.4- and PM_0.4–2.1-bound elements were moderate-to-strongly positively correlated with OP_v^DTT (r: 0.60–0.90). No significant correlation or dose–response relationship was found in PM_2.1–10. After respiratory exposure, several PM_0.4- and PM_0.4–2.1-bound elements had a moderate-to-strongly positive correlation with deposition fluxes of OP (defined as OP^Flux) (0.69–0.90). A generalized linear model analysis showed that the interquartile range (IQR) increase in the PM-bound elements (ng h⁻¹) was associated with a 41.7–58.1% increase in OP^Flux. Our study is a special case that enriches the knowledge of the association between OP^DTT and the chemical composition of PM of different sizes, especially after respiratory exposure, but the generalizability of the findings to other settings or types of PM may be limited. The associations among OP^DTT, other chemical compositions of PM, and human exposure risk merit further research. Full article

There is a need for monitoring air pollution associated with black carbon (BC) using a passive monitor is required in remote areas where the measurements are absent. In this pilot study, we developed a quantitative method to determine dry deposition submicron BC using dual-wavelength ultraviolet–visible spectroscopy. Furthermore, we measured the levels of dry deposition BC on plant leaves from 30 plant species located in urban Nanjing using the established method. The oxidative potential of BC on plant leaves as passive bio-monitoring samplers was assessed. The concentrations of black carbon (BC) on tree leaves varied from 0.01 to 1.6 mg m⁻². Significant differences in levels of BC across leaves from different tree types were observed. The values of oxidative potential in deposited particles of leaf samples were observed to be in the range of 33–46 nmol min⁻¹ mg⁻¹ using the dithiothreitol (DTT) assay and 18–32 nmol min⁻¹ mg⁻¹ using the ascorbic acid (AA) assay, respectively. In comparison, the oxidative potential of BC-dominated mass in water extracts of leaf samples was in the range of 5–35 nmol min⁻¹ mg⁻¹ measured using the DTT assay and 2 to 12 nmol min⁻¹ mg⁻¹ using the AA assay, respectively. We found variations in the levels of OP across the leaves of different tree types were not large, while the levels of OP in terms of BC-dominated mass varied greatly. These results indicate that the established method with dual-wavelength ultraviolet–visible spectroscopy could provide a simple tool to determine submicron BC in plant leaves of the passive monitor. Full article

Respiratory diseases have been proven to be directly related to air pollutants. Xuanwei, located in South China, has been known to have the highest mortality rate for lung cancer in China because of the air pollutants emitted through local coal combustion. However, the mechanism of lung cancer induced by air pollutants is not clear. Based on the fact that a large number of iron-bearing mineral particles was found in Xuanwei coal combustion particles, the iron-containing particles were hypothesized to play important roles in the pathogenesis of the high incidence rate of lung cancer in this area. In this study, raw coal samples were collected from a coal mine in the Xuanwei area. Size-resolved particles emitted from the raw coal samples were collected using an Anderson high-volume sampler. Mineralogical characterization and an assessment of the oxidative potential of the iron-containing particles were conducted using cutting-edge technologies, and the biological activity of the particles were evaluated via DTT assay. Our data showed that the iron-containing minerals accounted for more than 10% of the measured particles emitted from Xuanwei coal combustion samples. The content analysis of ·OH generated from Xuanwei coal combustion particles showed that ·OH content was dependent on the size of particles in the surrogated lung fluid. The concentration of ·OH increased as the particle size decreased. The DTT assay data further demonstrated that when the mass concentration of dissolved irons increased, the oxidation potential of the particles increased. The highest proportion of divalent iron in the total dissolved iron was found in the submicron particles in low pH solution(pH = 1), which indicated that the oxidative potential induced by submicron particles was stronger than that induced by coarse particles and fine particles. Armed with the above data, the toxicological mechanism of the iron-containing mineral particles can be investigated further. Full article

Selective delivery of anticancer drug molecules to the tumor site enhances local drug dosages, which leads to the death of cancer cells while simultaneously minimizing the negative effects of chemotherapy on other tissues, thereby improving the patient’s quality of life. To address this need, we developed reduction-responsive chitosan-based injectable hydrogels via the inverse electron demand Diels–Alder reaction between tetrazine groups of disulfide-based cross-linkers and norbornene groups of chitosan derivatives, which were applied to the controlled delivery of doxorubicin (DOX). The swelling ratio, gelation time (90–500 s), mechanical strength (G’~350–850 Pa), network morphology, and drug-loading efficiency (≥92%) of developed hydrogels were investigated. The in vitro release studies of the DOX-loaded hydrogels were performed at pH 7.4 and 5.0 with and without DTT (10 mM). The biocompatibility of pure hydrogel and the in vitro anticancer activity of DOX-loaded hydrogels were demonstrated via MTT assay on HEK-293 and HT-29 cancer cell lines, respectively. Full article

Fine particulate matter (PM_2.5) is a major contributor to the degree of air pollution, and it is associated with a range of adverse health impacts. Moreover, the oxidative potential (OP, as a tracer of oxidative stress) of PM_2.5 has been thought to be a possible determinant of its health impact. In this study, the OP of 136 fine aerosol filter samples collected in Changzhou in two seasons (spring and summer) were determined using a dithiothreitol (DTT) assay. Source apportionments of the PM_2.5 and DTT activity were further performed. Our results showed that the daily average ± standard deviation of the DTT_v (volume-normalized DTT activity) in the PM_2.5 was 1.16 ± 0.58 nmol/min/m³ and 0.85 ± 0.16 nmol/min/m³ in the spring and summer, respectively, and the DTT_m (mass-normalized DTT activity) was 13.56 ± 5.45 pmol/min/μg and 19.97 ± 6.54 pmol/min/μg in the spring and summer, respectively. The DTT_v was higher in the spring compared to the summer while the opposite was true for the DTT_m. Most of the detected components (including the organic component, element component, NH₄⁺, Mn, Cu, Zn, etc.) exhibited a moderately positive correlation with the DTT_v, but the opposite was found with the DTT_m. An aerodyne high-resolution aerosol mass spectrometer (HP-AMS) was deployed to probe the chemical properties of the water-soluble organic matter (WSOA). Positive matrix factorization (PMF) coupled with multiple linear regression was used to obtain the relative source contributions to the DTT activity for the WSOA in the PM_2.5. The results showed that the sensitivity sequences of the DTT_v to the WSOA sources were oxygenated organic aerosol (OOA) > biomass burning OA (BBOA) > hydrocarbon-like OA (HOA) in the spring and HOA > nitrogen-enriched OA (NOA) > OOA in the summer. The PMF suggested the highest contribution from traffic emissions to the DTT_v of the PM_2.5 in both seasons. Our findings point to the importance of both organic components from secondary formation and transition metals to adverse health effects in this region. This study can provide an important reference for adopting appropriate public health policies regarding the detrimental outcomes of exposure to PM_2.5. Full article

The antioxidant activity and chemical stability of 6-amino-6-deoxy-L-ascorbic acid (D1) and N-methyl-6-amino-6-deoxy-L-ascorbic acid (D2) were examined with ABTS and DPPH assays and compared with the reference L-ascorbic acid (AA). In addition, the optimal storing conditions, as well as the pH at which the amino derivatives maintain stability, were determined using mass spectrometry. Comparable antioxidant activities were observed for NH-bioisosteres and AA. Moreover, D1 showed higher stability in an acidic medium than the parent AA. In addition, AA, D1, and D2 share the same docking profile, with wild-type human peroxiredoxin as a model system. Their docking scores are similar to those of dithiothreitol (DTT). This suggests a similar binding affinity to the human peroxiredoxin binding site. Full article

Throughout the cold and the warm periods of 2020, chemical and toxicological characterization of the water-soluble fraction of size segregated particulate matter (PM) (<0.49, 0.49–0.95, 0.95–1.5, 1.5–3.0, 3.0–7.2 and >7.2 μm) was conducted in the urban agglomeration of Thessaloniki, northern Greece. Chemical analysis of the water-soluble PM fraction included water-soluble organic carbon (WSOC), humic-like substances (HULIS), and trace elements (V, Cr, Mn, Fe, Ni, Cu, Zn, As, Cd and Pb). The bulk (sum of all size fractions) concentrations of HULIS were 2.5 ± 0.5 and 1.2 ± 0.3 μg m⁻³, for the cold and warm sampling periods, respectively with highest values in the <0.49 μm particle size fraction. The total HULIS-C/WSOC ratio ranged from 17 to 26% for all sampling periods, confirming that HULIS are a significant part of WSOC. The most abundant water-soluble metals were Fe, Zn, Cu, and Mn. The oxidative PM activity was measured abiotically using the dithiothreitol (DTT) assay. In vitro cytotoxic responses were investigated using mitochondrial dehydrogenase (MTT). A significant positive correlation was found between OP_m^DTT, WSOC, HULIS and the MTT cytotoxicity of PM. Multiple Linear Regression (MLR) showed a good relationship between OP_M^DTT, HULIS and Cu. Full article

P21-activated kinase 1 (PAK1), as a member of the PAK family, has been implicated in various functions during somatic mitosis; however, less is known about its role during oocyte meiosis. Herein, we highlight the indispensable role of PAK1 in regulating spindle assembly and cell cycle progression during the first meiotic division of porcine oocytes. First, we found that the activated PAK1 expressed dynamically, and its subcellular localization was tightly associated with the spindle dynamics during meiosis in porcine oocytes. Specific inhibition of PAK1 activity by inhibitor targeting PAK1 activation-3 (IPA-3) led to impaired extrusion of the first polar body (PB1); with most of the IPA-3-treated oocytes arrested at germinal vesicle breakdown (GVBD) and subjected to failure of bipolar spindle formation. However, the adverse effects caused by IPA-3 on oocytes could be restored by reducing disulfide bonds between PAK1 and IPA-3 with dithiothreitol (DTT) treatment. Furthermore, the co-immunoprecipitation assay revealed that PAK1 interacted directly with Aurora A and transforming acidic coiled coil 3 (TACC3), providing an additional explanation for the similar localization of Aurora A and activated PAK1. Additionally, inhibiting the activity of PAK1 decreased the expression of p-Aurora A and p-TACC3; however, the reduced activity of Aurora A and TACC3 could be restored by DTT. In conclusion, PAK1 plays a crucial role in the proper assembly of the spindle during the first meiotic division of porcine oocytes, and the regulation of PAK1 is associated with its effects on p-Aurora A and p-TACC3 expression. Full article

The present study describes the method of preparing reagents containing firefly luciferase (FLuc) and its substrate, D-luciferin, immobilized into gelatin gel separately or together. The addition of stabilizers dithiothreitol (DTT) and bovine serum albumin (BSA) to the reagent is a factor in achieving higher activity of reagents and their stability during storage. The use of immobilized reagents substantially simplifies the procedure of assay for microbial contamination. The mechanism of action of the reagents is based on the relationship between the intensity of the bioluminescent signal and the level of ATP contained in the solution of the lysed bacterial cells. The highest sensitivity to ATP is achieved by using immobilized FLuc or reagents containing separately immobilized FLuc and D-luciferase. The limit of detection of ATP by the developed reagents is 0.3 pM, which corresponds to 20,000 cells·mL⁻¹. The linear response range is between 0.3 pM and 3 nM ATP. The multicomponent reagent, containing co-immobilized FLuc and D-luciferin, shows insignificantly lower sensitivity to ATP—0.6 pM. Moreover, the proposed method of producing an immobilized firefly luciferin-luciferase system holds considerable promise for the development of bioluminescent biosensors intended for the analysis of microbial contamination. Full article

Gasoline emissions contain high levels of pollutants, including particulate matter (PM), which are associated with several health outcomes. Moreover, due to the depletion of fossil fuels, biofuels represent an attractive alternative, particularly second-generation biofuels (B2G) derived from lignocellulosic biomass. Unfortunately, compared to the abundant literature on diesel and gasoline emissions, relatively few studies are devoted to alternative fuels and their health effects. This study aimed to compare the adverse effects of gasoline and B2G emissions on human bronchial epithelial cells. We characterized the emissions generated by propane combustion (CAST1), gasoline Surrogate, and B2G consisting of Surrogate blended with anisole (10%) (S+10A) or ethanol (10%) (S+10E). To study the cellular effects, BEAS-2B cells were cultured at air-liquid interface for seven days and exposed to different emissions. Cell viability, oxidative stress, inflammation, and xenobiotic metabolism were measured. mRNA expression analysis was significantly modified by the Surrogate S+10A and S+10E emissions, especially CYP1A1 and CYP1B1. Inflammation markers, IL-6 and IL-8, were mainly downregulated doubtless due to the PAHs content on PM. Overall, these results demonstrated that ultrafine particles generated from biofuels Surrogates had a toxic effect at least similar to that observed with a gasoline substitute (Surrogate), involving probably different toxicity pathways. Full article