Synergistic and Antagonistic Interactions among the Particulate Matter Components in Generating Reactive Oxygen Species Based on the Dithiothreitol Assay

We assessed the interactions among the particulate matter (PM) components in generating the reactive oxygen species (ROS) based on a dithiothreitol (DTT) assay. We started with the standard solutions of known redox-active substances, i.e., quinones (9,10-phenanthraquinone, 1,2-naphthoquinone, 1,4-naphthoquinone, and 5-hydroxy-1,4-naphthoquinone) and metals [Fe (II), Mn (II), and Cu (II)]. Both DTT consumption and hydroxyl radical (·OH) generation were measured in the DTT assay. The interactions of Fe were additive with quinones in DTT consumption but strongly synergistic in ·OH generation. Cu showed antagonistic interactions with quinones in both DTT consumption and ·OH generation. Mn interacted synergistically with quinones in DTT oxidation but antagonistically in ·OH generation. The nature of the interactions of these metals (Fe, Mn, and Cu) with ambient humic-like substances (HULIS) resembled that with quinones, although the intensity of interactions were weaker in DTT consumption than ·OH generation. Finally, we demonstrated that the DTT consumption capability of ambient PM can be well explained by HULIS, three transition metals (Fe, Mn, and Cu), and their interactions, but ·OH generation involves a contribution (∼50%) from additional compounds (aliphatic species or metals other than Fe, Mn, and Cu) present in the hydrophilic PM fraction. The study highlights the need to account for the interactions between organic compounds and metals, while apportioning the relative contributions of chemical components in the PM oxidative potential

Rethinking Dithiothreitol-Based Particulate Matter Oxidative Potential: Measuring Dithiothreitol Consumption versus Reactive Oxygen Species Generation

We measured the rate of generation of reactive oxygen species (ROS) [hydroxyl radicals (^<b>•</b>OH) and hydrogen peroxide (H₂O₂)] catalyzed by ambient particulate matter (PM) in the dithiothreitol (DTT) assay. To understand the mechanism of ROS generation, we tested several redox-active substances, such as 9,10-phenanthrenequinone (PQ), 5-hydroxy-1,4-naphthoquinone (5H-1,4NQ), 1,2-naphthoquinone (1,2-NQ), 1,4-naphthoquinone (1,4-NQ), copper­(II), manganese­(II), and iron (II and III). Both pure compounds and their mixtures show different patterns in DTT oxidation versus ROS generation. The quinones, known to oxidize DTT in the efficiency order of PQ > 5H-1,4NQ > 1,2-NQ > 1,4-NQ, show a different efficiency order (5H-1,4NQ > 1,2-NQ ≈ PQ > 1,4-NQ) in the ROS generation. Cu­(II), a dominant metal in DTT oxidation, contributes almost negligibly to the ROS generation. Fe is mostly inactive in DTT oxidation, but shows synergistic effect in ^<b>•</b>OH formation in the presence of other quinones (mixture/sum > 1.5). Ten ambient PM samples collected from an urban site were analyzed, and although DTT oxidation was significantly correlated with H₂O₂ generation (Pearson’s <i>r</i> = 0.91), no correlation was observed between DTT oxidation and ^<b>•</b>OH formation. Our results show that measuring both DTT consumption and ROS generation in the DTT assay is important to incorporate the synergistic contribution from different aerosol components and to provide a more inclusive picture of the ROS activity of ambient PM

Ambient Size Distributions and Lung Deposition of Aerosol Dithiothreitol-Measured Oxidative Potential: Contrast between Soluble and Insoluble Particles

Ambient particulate matter may upset redox homeo­stasis, leading to oxidative stress and adverse health effects. Size distributions of water-insoluble and water-soluble OP^DTT (dithio­threitol assay, measure of oxidative potential per air volume) are reported for a road­side site and an urban site. The average water-insoluble fractions were 23% and 51%, and 37% and 39%, for fine and coarse modes at the roadside and urban sites, respectively, measured during different periods. Water-soluble OP^DTT was unimodal, peaked near 1–2.5 μm due to contributions from fine-mode organic components plus coarse-mode transition metal ions. In contrast, water-insoluble OP^DTT was bimodal, with both fine and coarse modes. The main chemical components that drive both fractions appear to be the same, except that for water-insoluble OP^DTT the compounds were absorbed on surfaces of soot and non-tailpipe traffic dust. They were largely externally mixed and deposited in different regions in the respiratory system, transition metal ions predominately in the upper regions and organic species, such as quinones, deeper in the lung. Although OP^DTT per mass (toxicity) was highest for ultrafine particles, estimated lung deposition was mainly from accumulation and coarse particles. Contrasts in the phases of these forms of OP^DTT deposited in the respiratory system may have differing health impacts

Contribution of Water-Soluble and Insoluble Components and Their Hydrophobic/Hydrophilic Subfractions to the Reactive Oxygen Species-Generating Potential of Fine Ambient Aerosols

Relative contributions of water- and methanol-soluble compounds and their hydrophobic/hydrophilic subfractions to the ROS (reactive oxygen species)-generating potential of ambient fine aerosols (<i>D</i>_p < 2.5 μm) are assessed. ROS-generating (or oxidative) potential of the particulate matter (PM) was measured by the dithiothreitol (DTT) assay. Particles were collected on quartz filters (<i>N</i> = 8) at an urban site near central Atlanta during January–February 2012 using a PM_2.5 high-volume sampler. Filter punches were extracted separately in both water and methanol. Hydrophobic and hydrophilic fractions were then subsequently segregated via a C-18 solid phase extraction column. The DTT assay response was significantly higher for the methanol extract, and for both extracts a substantial fraction of PM oxidative potential was associated with the hydrophobic compounds as evident from a substantial attenuation in DTT response after passing PM extracts through the C-18 column (64% for water and 83% for methanol extract; both median values). The DTT activities of water and methanol extracts were correlated with the water-soluble (<i>R</i> = 0.86) and water-insoluble organic carbon (<i>R</i> = 0.94) contents of the PM, respectively. Brown carbon (BrC), which predominantly represents the hydrophobic organic fraction (referred to as humic-like substances, HULIS), was also correlated with DTT activity in both the water (<i>R</i> = 0.78) and methanol extracts (<i>R</i> = 0.83). Oxidative potential was not correlated with any metals measured in the extracts. These findings suggest that the hydrophobic components of both water-soluble and insoluble organic aerosols substantially contribute to the oxidative properties of ambient PM. Further investigation of these hydrophobic organic compounds could help identify sources of a significant fraction of ambient aerosol toxicity

Organic Aerosols Associated with the Generation of Reactive Oxygen Species (ROS) by Water-Soluble PM_2.5

We compare the relative toxicity of various organic aerosol (OA) components identified by an aerosol mass spectrometer (AMS) based on their ability to generate reactive oxygen species (ROS). Ambient fine aerosols were collected from urban (three in Atlanta, GA and one in Birmingham, AL) and rural (Yorkville, GA and Centerville, AL) sites in the Southeastern United States. The ROS generating capability of the water-soluble fraction of the particles was measured by the dithiothreitol (DTT) assay. Water-soluble PM extracts were further separated into the hydrophobic and hydrophilic fractions using a C-18 column, and both fractions were analyzed for DTT activity and water-soluble metals. Organic aerosol composition was measured at selected sites using a high-resolution time-of-flight AMS. Positive matrix factorization of the AMS spectra resolved the organic aerosol into isoprene-derived OA (Isop_OA), hydrocarbon-like OA (HOA), less-oxidized oxygenated OA, (LO-OOA), more-oxidized OOA (MO-OOA), cooking OA (COA), and biomass burning OA (BBOA). The association of the DTT activity of water-soluble PM_2.5 (WS_DTT) with these factors was investigated by linear regression techniques. BBOA and MO-OOA were most consistently linked with WS_DTT, with intrinsic water-soluble activities of 151 ± 20 and 36 ± 22 pmol/min/μg, respectively. Although less toxic, MO-OOA was most widespread, contributing to WS_DTT activity at all sites and during all seasons. WS_DTT activity was least associated with biogenic secondary organic aerosol. The OA components contributing to WS_DTT were humic-like substances (HULIS), which are abundantly emitted in biomass burning (BBOA) and include highly oxidized OA from multiple sources (MO-OOA). Overall, OA contributed approximately 60% to the WS_DTT activity, with the remaining probably from water-soluble metals, which were mostly associated with the hydrophilic WS_DTT fraction

Microstructural details of the eggshells.

(A) Stereoscopic binocular microscope photomicrograph of the tangential image of eggshell from clutch P32 showing compactituberculate ornamentation in the form of dense nodes separated by pore spaces (see arrow) (Scale bar: 2 mm). (B) Radial thin section of eggshell oospecies Megaloolithus cylindricus from clutch A3 showing long and cylindrical shell units with few areas of diagenetic alteration and vertical tubocanaliculate pore canals (see white arrow). The growth lines are limited to individual shell units (after Dhiman et al. [15]). (C) Radial thin section of M. jabalpurensis eggshell from clutch P11 showing fan-shaped shell units and arching growth lines. (D) Highly altered radial thin section of the oospecies M. dhoridungriensis from clutch P47 showing conical shell units which are broader in the upper part. (E) Radial thin section of eggshell representing oospecies F. mohabeyi from clutch P22 showing fused shell units and growth lines merging with each other (after Dhiman et al. [15]). (F) Radial thin section of F. baghensis eggshells from clutch J9 showing fused shell units and swollen basal end units. (G) Radial thin section of F. padiyalensis eggshells from clutch A5 showing long shell units fusing with each other in the lower parts (Scale bar from (B) to (G): 1000 μm). (H) SEM photograph of tangential surface of eggshell from clutch DR8 showing resorption craters (see arrow).</p

Field photographs showing sedimentary features in the clutch-bearing outcrops.

(A) The outcrop from Dholiya Raipuriya shows a characteristic brecciated nodular structure. Some nodules show autobrecciation represented as brecciated fragments with a gap in between them which indicates that the nodules fitted with each other before the disruption. The autobrecciation indicates the non-transported character of the nodules. The areas where zig-saw fit does not exist between nodules indicate their rotation and translocation after shrinkage and collapse (after Dhiman et al. [15]). (B) Intraclast collapse breccias from Dholiya Raipuriya showing variably spaced sub-angular, sub-rounded, and elliptical shaped coarse-grained brecciated clasts in a carbonate matrix. (C) In outcrops at Padlya, the chert exists in association with brecciated nodular limestone where the light grey zones show shrinkage characteristics while the dark grey zones are matrix-rich areas. The brecciated nodules also show zig-saw fit at some places while at other areas the zig-saw fit has collapsed.</p

Schematic diagram of clutch P1 from Padlya, M.P. showing evidence for 20 eggs.

The eggs i to xii show closely grouped eggs while other eggs in the clutch (xviii to xx) are spaced at a distance from these grouped eggs.</p

Reactive Oxygen Species Generation Linked to Sources of Atmospheric Particulate Matter and Cardiorespiratory Effects

Exposure to atmospheric fine particulate matter (PM_2.5) is associated with cardiorespiratory morbidity and mortality, but the mechanisms are not well understood. We assess the hypothesis that PM_2.5 induces oxidative stress in the body via catalytic generation of reactive oxygen species (ROS). A dithiothreitol (DTT) assay was used to measure the ROS-generation potential of water-soluble PM_2.5. Source apportionment on ambient (Atlanta, GA) PM_2.5 was performed using the chemical mass balance method with ensemble-averaged source impact profiles. Linear regression analysis was used to relate PM_2.5 emission sources to ROS-generation potential and to estimate historical levels of DTT activity for use in an epidemiologic analysis for the period of 1998–2009. Light-duty gasoline vehicles (LDGV) exhibited the highest intrinsic DTT activity, followed by biomass burning (BURN) and heavy-duty diesel vehicles (HDDV) (0.11 ± 0.02, 0.069 ± 0.02, and 0.052 ± 0.01 nmol min^–1 μg^–1_source, respectively). BURN contributed the largest fraction to total DTT activity over the study period, followed by LDGV and HDDV (45, 20, and 14%, respectively). DTT activity was more strongly associated with emergency department visits for asthma/wheezing and congestive heart failure than PM_2.5. This work provides further epidemiologic evidence of a biologically plausible mechanism, that of oxidative stress, for associations of adverse health outcomes with PM_2.5 mass and supports continued assessment of the utility of the DTT activity assay as a measure of ROS-generating potential of particles

Discovery of Peptidomimetic Antibody–Drug Conjugate Linkers with Enhanced Protease Specificity

Antibody–drug conjugates (ADCs) have become an important therapeutic modality for oncology, with three approved by the FDA and over 60 others in clinical trials. Despite the progress, improvements in ADC therapeutic index are desired. Peptide-based ADC linkers that are cleaved by lysosomal proteases have shown sufficient stability in serum and effective payload-release in targeted cells. If the linker can be preferentially hydrolyzed by tumor-specific proteases, safety margin may improve. However, the use of peptide-based linkers limits our ability to modulate protease specificity. Here we report the structure-guided discovery of novel, nonpeptidic ADC linkers. We show that a cyclobutane-1,1-dicarboxamide-containing linker is hydrolyzed predominantly by cathepsin B while the valine–citrulline dipeptide linker is not. ADCs bearing the nonpeptidic linker are as efficacious and stable in vivo as those with the dipeptide linker. Our results strongly support the application of the peptidomimetic linker and present new opportunities for improving the selectivity of ADCs