Activity-Based Protein Profiling to Probe Relationships between Cytochrome P450 Enzymes and Early-Age Metabolism of Two Polycyclic Aromatic Hydrocarbons (PAHs): Phenanthrene and Retene.

A growing body of literature has linked early-life exposures to polycyclic aromatic hydrocarbons (PAH) with adverse neurodevelopmental effects. Once in the body, metabolism serves as a powerful mediator of PAH toxicity by bioactivating and detoxifying PAH metabolites. Since enzyme expression and activity vary considerably throughout human development, we evaluated infant metabolism of PAHs as a potential contributing factor to PAH susceptibility. We measured and compared rates of phenanthrene and retene (two primary PAH constituents of woodsmoke) metabolism in human hepatic microsomes from individuals ≤21 months of age to a pooled sample (n = 200) consisting primarily of adults. We used activity-based protein profiling (ABPP) to characterize cytochrome P450 enzymes (CYPs) in the same hepatic microsome samples. Once incubated in microsomes, phenanthrene demonstrated rapid depletion. Best-fit models for phenanthrene metabolism demonstrated either 1 or 2 phases, depending on the sample, indicating that multiple enzymes could metabolize phenanthrene. We observed no statistically significant differences in phenanthrene metabolism as a function of age, although samples from the youngest individuals had the slowest phenanthrene metabolism rates. We observed slower rates of retene metabolism compared with phenanthrene also in multiple phases. Rates of retene metabolism increased in an age-dependent manner until adult (pooled) metabolism rates were achieved at ∼12 months. ABPP identified 28 unique CYPs among all samples, and we observed lower amounts of active CYPs in individuals ≤21 months of age compared to the pooled sample. Phenanthrene metabolism correlated to CYPs 1A1, 1A2, 2C8, 4A22, 3A4, and 3A43 and retene metabolism correlated to CYPs 1A1, 1A2, and 2C8 measured by ABPP and vendor-supplied substrate marker activities. These results will aid efforts to determine human health risk and susceptibility to PAHs exposure during early life.

Stereoselective Single Step Cyclization to Give Belt-Functionalized Pillar[6]arenes.

Two macrocycles were synthesized through cyclization reactions of secondary benzylic alcohols, giving pillar[6]arenes with a methyl substituent at each belt position. These macrocycles form stereoselectively with only the rtctct isomer with alternating up and down orientations of the belt methyl groups definitively identified. Isolated yields were modest (7 and 9%), but the macrocycles are prepared in a single step from either a commercially available alcohol or a very readily prepared precursor. X-ray crystal structures of the macrocycles indicate they have a capsule-like structure, which is far from the conventional pillar shape. Density functional theory calculations reveal that the energy barrier required to obtain the pillar conformation is significantly higher for these belt-functionalized macrocycles than for conventional belt-unfunctionalized pillar[6]arenes.

Benzo[a]pyrene toxicokinetics in humans following dietary supplementation with 3,3'-diindolylmethane (DIM) or Brussels sprouts.

Utilizing the atto-zeptomole sensitivity of UPLC-accelerator mass spectrometry (UPLC-AMS), we previously demonstrated significant first-pass metabolism following escalating (25-250 ng) oral micro-dosing in humans of [14C]-benzo[a]pyrene ([14C]-BaP). The present study examines the potential for supplementation with Brussels sprouts (BS) or 3,3'-diindolylmethane (DIM) to alter plasma levels of [14C]-BaP and metabolites over a 48-h period following micro-dosing with 50 ng (5.4 nCi) [14C]-BaP. Volunteers were dosed with [14C]-BaP following fourteen days on a cruciferous vegetable restricted diet, or the same diet supplemented for seven days with 50 g of BS or 300 mg of BR-DIM® prior to dosing. BS or DIM reduced total [14C] recovered from plasma by 56-67% relative to non-intervention. Dietary supplementation with DIM markedly increased Tmax and reduced Cmax for [14C]-BaP indicative of slower absorption. Both dietary treatments significantly reduced Cmax values of four downstream BaP metabolites, consistent with delaying BaP absorption. Dietary treatments also appeared to reduce the T1/2 and the plasma AUC(0,∞) for Unknown Metabolite C, indicating some effect in accelerating clearance of this metabolite. Toxicokinetic constants for other metabolites followed the pattern for [14C]-BaP (metabolite profiles remained relatively consistent) and non-compartmental analysis did not indicate other significant alterations. Significant amounts of metabolites in plasma were at the bay region of [14C]-BaP irrespective of treatment. Although the number of subjects and large interindividual variation are limitations of this study, it represents the first human trial showing dietary intervention altering toxicokinetics of a defined dose of a known human carcinogen.

Balancing on a Knife's Edge: Studies on the Synthesis of Pillar[6]arene Derivatives.

Pillar[6]arenes are established as crucial building blocks in supramolecular chemistry; however, they can be difficult to synthesize, particularly in the absence of large solubilizing substituents. In this work, we explore variability in literature syntheses of pillar[6]arene derivatives and suggest that the outcome is dependent on whether oligomeric intermediates stay in solution long enough for the thermodynamically favorable macrocyclization to occur. We demonstrate that in a previously capricious BF3·OEt2-mediated procedure, ≤5 mol % of a Brønsted acid can slow down the reaction to favor macrocycle formation.

Exploring the Excited States of a Hexa-peri-hexabenzocoronene-Substituted Dipyridophenazine Ligand and Its Metal Complexes.

A hexa-peri-hexabenzocoronene (HBC)-substituted dipyridophenazine (dppz) ligand (dppz-HBC) and its corresponding rhenium [Re(CO)3Cl] and ruthenium [Ru(bpy)2]2+ complexes were synthesized and characterized. The interplay of their various excited states was investigated using spectroscopic and computational techniques. Perturbation of the HBC was seen through a broadening and decreased intensity of the HBC absorption bands that dominate the absorption spectra. A delocalized, partial charge transfer state was shown through emission (520 nm) in the ligand and rhenium complex and is supported by time-dependent density functional theory calculations. Transient absorption measurements revealed the presence of dark states with a triplet delocalized state populated in the ligand, while in the complexes, longer-lived (2.3-2.5 µs) triplet HBC states could be accessed. The properties of the studied ligand and complexes provide insight into the future design of polyaromatic systems and add to the rich history of dppz systems.

Translating dosimetry of Dibenzo[def,p]chrysene (DBC) and metabolites across dose and species using physiologically based pharmacokinetic (PBPK) modeling.

Dibenzo[def,p]chrysene (DBC) is an environmental polycyclic aromatic hydrocarbon (PAH) that causes tumors in mice and has been classified as a probable human carcinogen by the International Agency for Research on Cancer. Animal toxicity studies often utilize higher doses than are found in relevant human exposures. Additionally, like many PAHs, DBC requires metabolic bioactivation to form the ultimate toxicant, and species differences in DBC and DBC metabolite metabolism have been observed. To understand the implications of dose and species differences, a physiologically based pharmacokinetic model (PBPK) for DBC and major metabolites was developed in mice and humans. Metabolism parameters used in the model were obtained from experimental in vitro metabolism assays using mice and human hepatic microsomes. PBPK model simulations were evaluated against mice dosed with 15 mg/kg DBC by oral gavage and human volunteers orally microdosed with 29 ng of DBC. DBC and its primary metabolite DBC-11,12-diol were measured in blood of mice and humans, while in urine, the majority of DBC metabolites were obeserved as conjugated DBC-11,12-diol, conjugated DBC tetrols, and unconjugated DBC tetrols. The PBPK model was able to predict the time course concentrations of DBC, DBC-11,12-diol, and other DBC metabolites in blood and urine of human volunteers and mice with reasonable accuracy. Agreement between model simulations and measured pharmacokinetic data in mice and human studies demonstrate the success and versatility of our model for interspecies extrapolation and applicability for different doses. Furthermore, our simulations show that internal dose metrics used for risk assessment do not necessarily scale allometrically, and that PBPK modeling provides a reliable approach to appropriately account for interspecies differences in metabolism and physiology.

Profiling How the Gut Microbiome Modulates Host Xenobiotic Metabolism in Response to Benzo[a]pyrene and 1-Nitropyrene Exposure.

The gut microbiome is a key contributor to xenobiotic metabolism. Polycyclic aromatic hydrocarbons (PAHs) are an abundant class of environmental contaminants that have varying levels of carcinogenicity depending on their individual structures. Little is known about how the gut microbiome affects the rates of PAH metabolism. This study sought to determine the role that the gut microbiome has in determining the various aspects of metabolism in the liver, before and after exposure to two structurally different PAHs, benzo[a]pyrene and 1-nitropyrene. Following exposures, the metabolic rates of PAH metabolism were measured, and activity-based protein profiling was performed. We observed differences in PAH metabolism rates between germ-free and conventional mice under both unexposed and exposed conditions. Our activity-based protein profiling (ABPP) analysis showed that, under unexposed conditions, there were only minor differences in total P450 activity in germ-free mice relative to conventional mice. However, we observed distinct activity profiles in response to corn oil vehicle and PAH treatment, primarily in the case of 1-NP treatment. This study revealed that the repertoire of active P450s in the liver is impacted by the presence of the gut microbiome, which modifies PAH metabolism in a substrate-specific fashion.

Smartphone-Based Dual-Channel Immunochromatographic Test Strip with Polymer Quantum Dot Labels for Simultaneous Detection of Cypermethrin and 3-Phenoxybenzoic Acid.

Currently, gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-MS (LC-MS) are the primary methods used to detect pesticides and their metabolites for biomonitoring of exposure. Although GC-MS and LC-MS can provide accurate and sensitive measurements, these techniques are not suitable for point-of-care or in-field biomonitoring applications. The objective of this work is to develop a smartphone-based dual-channel immunochromatographic test strip (ICTS) for on-site biomonitoring of exposure to cypermethrin by simultaneous detection of cypermethrin and its metabolite, 3-phenoxybenzoic acid (3-PBA). Polymer carbon dots (PCDs) with ultrahigh fluorescent brightness were synthesized and used as a signal amplifier in ICTS assay. Cypermethrin (a representative pyrethroid pesticide) and its major metabolite 3-PBA were simultaneously detected to provide more comprehensive analysis of cypermethrin exposure. After competitive immunoreactions between the target sample and the coating antigens preloaded on the test line, the tracer antibody (PCD-conjugated antibody) was quantitatively captured on the test lines. The captured PCDs were inversely proportional to the amount of the target compound in the sample. The red fluorescence on the test line was then recorded using a smartphone-based device capable of conducting image analysis and recording. Under optimal conditions, the sensor showed excellent linear responses for detecting cypermethrin and 3-PBA ranging from 1 to 100 ng/mL and from 0.1 to 100 ng/mL, respectively, and the limits of detection were calculated to be ∼0.35 ng/mL for cypermethrin and ∼0.04 ng/mL for 3-PBA. The results demonstrate that the ICTS device is promising for accurate point-of-care biomonitoring of pesticide exposure.

3,3'-Diindolylmethane Exhibits Significant Metabolism after Oral Dosing in Humans.

3,3'-Diindolylmethane (DIM), a major phytochemical derived from ingestion of cruciferous vegetables, is also a dietary supplement. In preclinical models, DIM is an effective cancer chemopreventive agent and has been studied in a number of clinical trials. Previous pharmacokinetic studies in preclinical and clinical models have not reported DIM metabolites in plasma or urine after oral dosing, and the pharmacological actions of DIM on target tissues is assumed to be solely via the parent compound. Seven subjects (6 males and 1 female) ranging from 26-65 years of age, on a cruciferous vegetable-restricted diet prior to and during the study, took 2 BioResponse DIM 150-mg capsules (45.3 mg DIM/capsule) every evening for one week with a final dose the morning of the first blood draw. A complete time course was performed with plasma and urine collected over 48 hours and analyzed by UPLC-MS/MS. In addition to parent DIM, two monohydroxylated metabolites and 1 dihydroxylated metabolite, along with their sulfate and glucuronide conjugates, were present in both plasma and urine. Results reported here are indicative of significant phase 1 and phase 2 metabolism and differ from previous pharmacokinetic studies in rodents and humans, which reported only parent DIM present after oral administration. 3-((1H-indole-3-yl)methyl)indolin-2-one, identified as one of the monohydroxylated products, exhibited greater potency and efficacy as an aryl hydrocarbon receptor agonist when tested in a xenobiotic response element-luciferase reporter assay using Hepa1 cells. In addition to competitive phytochemical-drug adverse reactions, additional metabolites may exhibit pharmacological activity highlighting the importance of further characterization of DIM metabolism in humans. SIGNIFICANCE STATEMENT: 3,3'-Diindolylmethane (DIM), derived from indole-3-carbinol in cruciferous vegetables, is an effective cancer chemopreventive agent in preclinical models and a popular dietary supplement currently in clinical trials. Pharmacokinetic studies to date have found little or no metabolites of DIM in plasma or urine. In marked contrast, we demonstrate rapid appearance of mono- and dihydroxylated metabolites in human plasma and urine as well as their sulfate and glucuronide conjugates. The 3-((1H-indole-3-yl)methyl)indolin-2-one metabolite exhibited significant aryl hydrocarbon receptor agonist activity, emphasizing the need for further characterization of the pharmacological properties of DIM metabolites.

Exposure to an Environmental Mixture of Polycyclic Aromatic Hydrocarbons Induces Hepatic Cytochrome P450 Enzymes in Mice.

Cytochrome P450 enzymes (CYPs) play an important role in bioactivating or detoxifying polycyclic aromatic hydrocarbons (PAHs), common environmental contaminants. While it is widely accepted that exposure to PAHs induces CYPs, effectively increasing rates of xenobiotic metabolism, dose- and time-response patterns of CYP induction are not well-known. In order to better understand dose- and time-response relationships of individual CYPs following induction, we exposed B6129SF1/J mice to single or repeated doses (2-180 µmol/kg/d) of benzo[a]pyrene (BaP) or Supermix-10, a mixture of the top 10 most abundant PAHs found at the Portland Harbor Superfund Site. In hepatic microsomes from exposed mice, we measured amounts of active CYPs using activity-based protein profiling and total CYP expression using global proteomics. We observed rapid Cyp1a1 induction after 6 h at the lowest PAH exposures and broad induction of many CYPs after 3 daily PAH doses at 72 h following the first dose. Using samples displaying Cyp1a1 induction, we observed significantly higher metabolic affinity for BaP metabolism (Km reduced 3-fold), 3-fold higher intrinsic clearance, but no changes to the Vmax. Mice dosed with the highest PAH exposures exhibited 1.7-5-fold higher intrinsic clearance rates for BaP compared to controls and higher Vmax values indicating greater amounts of enzymes capable of metabolizing BaP. This study demonstrates exposure to PAHs found at superfund sites induces enzymes in dose- and time-dependent patterns in mice. Accounting for specific changes in enzyme profiles, relative rates of PAH bioactivation and detoxification, and resulting risk will help translate internal dosimetry of animal models to humans and improve risk assessments of PAHs at superfund sites.

Risk assessment of predicted serum concentrations of bisphenol A in children and adults following treatment with dental composite restoratives, dental sealants, or orthodontic adhesives using physiologically based pharmacokinetic modeling.

Bisphenol A (BPA) is a chemical used to manufacture bisphenol A glycidyl methacrylate (BisGMA). BisGMA has been used for decades in dental composite restoratives, sealants, and adhesives. Based on published studies, exposure to low concentrations of BPA are possible from dental and orthodontic devices. The serum BPA concentrations arising from such devices and oral doses were predicted using a PBPK model in children and adult females based on 1) published extraction data for cured and uncured 3M ESPE Filtek Supreme Ultra Flowable, 3M ESPE Filtek Bulk Fill Restorative, and 3M ESPE Clinpro Sealant and 2) published 20% ethanol/water and water rinsate data following orthodontic application with 3M ESPE Transbond MIP Primer and 3M ESPE Transbond XT Adhesive. Predicted oral exposure to BPA arising from these dental and orthodontic devices is low (median <10 ng/treatment) and predicted serum BPA concentrations were also low (<10-4 nM). Even the maximum predicted exposure in this study (533.2 ng/treatment) yields a margin of exposure of 7.5 relative to the EFSA t-TDI (4 µg/kg-day) and is only 2.8% of the daily BPA exposure for the US population in a 58-kg woman (15,660 ng/day). Therefore, the exposure to BPA arising from the 3M ESPE dental and orthodontic devices evaluated in this study is negligible relative to daily BPA exposure in the general population and these potential BPA sources do not constitute a risk to patients.

Structure Dependent Determination of Organophosphate Targets in Mammalian Tissues Using Activity-Based Protein Profiling.

Acute and chronic exposures to organophosphates (OPs), including agricultural pesticides, industrial chemicals, and chemical warfare agents, remain a significant worldwide health risk. The mechanisms by which OPs alter development and cognition in exposed individuals remain poorly understood, in part due to the large number of structurally diverse OPs and the wide range of affected proteins and signaling pathways. To investigate the influence of structure on OP targets in mammalian systems, we have developed a series of probes for activity-based protein profiling (ABPP) featuring two distinct reactive groups that mimic OP chemical reactivity. FOP features a fluorophosphonate moiety, and PODA and CODA utilize a dialkynyl phosphate ester; both reactive group types target serine hydrolase activity. As the oxon represents the highly reactive and toxic functional group of many OPs, the new probes described herein enhance our understanding of tissue-specific reactivity of OPs. Chemoproteomic analysis of mouse tissues treated with the probes revealed divergent protein profiles, demonstrating the influence of probe structure on protein targeting. These targets also vary in sensitivity toward different OPs. The simultaneous use of multiple probes in ABPP experiments may therefore offer more comprehensive coverage of OP targets; FOP consistently labeled more targets in both brain and liver than PODA or CODA, suggesting the dialkyne warhead is more selective for enzymes in major signaling pathways than the more reactive fluorophosphonate warhead. Additionally, the probes can be used to assess reactivation of OP-inhibited enzymes by N-oximes and may serve as diagnostic tools for screening of therapeutic candidates in a panel of protein targets. These applications will help clarify the short- and long-term effects of OP toxicity beyond acetylcholinesterase inhibition, investigate potential points of convergence for broad spectrum therapeutic development, and support future efforts to screen candidate molecules for efficacy in various model systems.

One-Step Synthesis of C2v-Symmetric Resorcin[4]arene Tetraethers.

The three-component reaction between a resorcinol, 1,3-dimethoxybenzene, and an alkyl aldehyde (R = C1-C11) along with BF3·OEt2 affords a C2v-symmetric resorcin[4]arene tetraether in one step; in most cases, the single isomer can be precipitated from the reaction mixture in moderate to excellent yields (up to 89%). The reaction is tolerant of 2-substituted resorcinols (R' = OH, Cl, Br, Me), allowing a third type of functionality to be regioselectively incorporated during the macrocyclization.

Spatially Resolved Proteome Mapping of Laser Capture Microdissected Tissue with Automated Sample Transfer to Nanodroplets.

Current mass spectrometry (MS)-based proteomics approaches are ineffective for mapping protein expression in tissue sections with high spatial resolution because of the limited overall sensitivity of conventional workflows. Here we report an integrated and automated method to advance spatially resolved proteomics by seamlessly coupling laser capture microdissection (LCM) with a recently developed nanoliter-scale sample preparation system termed nanoPOTS (Nanodroplet Processing in One pot for Trace Samples). The workflow is enabled by prepopulating nanowells with DMSO, which serves as a sacrificial capture liquid for microdissected tissues. The DMSO droplets efficiently collect laser-pressure catapulted LCM tissues as small as 20 µm in diameter with success rates >87%. We also demonstrate that tissue treatment with DMSO can significantly improve proteome coverage, likely due to its ability to dissolve lipids from tissue and enhance protein extraction efficiency. The LCM-nanoPOTS platform was able to identify 180, 695, and 1827 protein groups on average from 12-µm-thick rat brain cortex tissue sections having diameters of 50, 100, and 200 µm, respectively. We also analyzed 100-µm-diameter sections corresponding to 10-18 cells from three different regions of rat brain and comparatively quantified ∼1000 proteins, demonstrating the potential utility for high-resolution spatially resolved mapping of protein expression in tissues.

Linking internal dosimetries of the propyl metabolic series in rats and humans using physiologically based pharmacokinetic (PBPK) modeling.

The metabolic series approach has successfully linked internal dosimetries of metabolically related compounds reducing cost and time for chemical risk assessments. Here, we developed a physiologically based pharmacokinetic (PBPK) model in rats and humans for the propyl metabolic series including propyl acetate, 1-propanol, propionaldehyde, and propionic acid. Manufacturers use these compounds as organic solvents and intermediates during chemical synthesis. Public exposures can occur through using consumer products containing propyl compounds like cosmetics, aerosol sprays, or foods, and occupational exposures can occur at manufacturing facilities. To develop the PBPK model, we measured in vitro metabolism of propyl acetate in blood and liver S9 fractions. We measured concentrations of propyl compounds in blood following intravenous (iv) infusion of 13C-propanol or 13C-propionic acid and closed chamber inhalation exposures to propyl acetate or propanol in rats. Using these studies and other published data, we modified an existing PBPK model for the butyl metabolic series to simulate time course concentrations of propyl compounds in rats and humans. Consistent with measured in vitro and in vivo data, the optimized propyl series model predicts rapid clearance of propyl acetate, higher concentrations of propanol in blood from propyl acetate inhalation compared to propanol inhalation in rats but not in humans, and low concentrations of propionic acid in blood from exposures to propyl acetate or propanol. Regulators can use this model as a tool for propyl compound risk assessment by linking internal dosimetries under various exposure scenarios.

Benzo[ a]pyrene Induction of Glutathione S-Transferases: An Activity-Based Protein Profiling Investigation.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants generated from combustion of carbon-based matter. Upon ingestion, these molecules can be bioactivated by cytochrome P450 monooxygenases to oxidized toxic metabolites. Some of these metabolites are potent carcinogens that can form irreversible adducts with DNA and other biological macromolecules. Conjugative enzymes, such as glutathione S-transferases or UDP-glucuronosyltransferases, are responsible for the detoxification and/or facilitate the elimination of these carcinogens. While responses to PAH exposures have been extensively studied for the bioactivating cytochrome P450 enzymes, much less is known regarding the response of glutathione S-transferases in mammalian systems. In this study, we investigated the expression and activity responses of murine hepatic glutathione S-transferases to benzo[ a]pyrene exposure using global proteomics and activity-based protein profiling for chemoproteomics, respectively. Using this approach, we identified several enzymes exhibiting increased activity including GSTA2, M1, M2, M4, M6, and P1. The activity of one GST enzyme, GSTA4, was found to be downregulated with increasing B[ a]P dose. Activity responses of several of these enzymes were identified as being expression-independent when comparing global and activity-based data sets, possibly alluding to as of yet unknown regulatory post-translational mechanisms.

Multifunctional Activity-Based Protein Profiling of the Developing Lung.

Lung diseases and disorders are a leading cause of death among infants. Many of these diseases and disorders are caused by premature birth and underdeveloped lungs. In addition to developmentally related disorders, the lungs are exposed to a variety of environmental contaminants and xenobiotics upon birth that can cause breathing issues and are progenitors of cancer. In order to gain a deeper understanding of the developing lung, we applied an activity-based chemoproteomics approach for the functional characterization of the xenometabolizing cytochrome P450 enzymes, active ATP and nucleotide binding enzymes, and serine hydrolases using a suite of activity-based probes (ABPs). We detected P450 activity primarily in the postnatal lung; using our ATP-ABP, we characterized a wide range of ATPases and other active nucleotide- and nucleic acid-binding enzymes involved in multiple facets of cellular metabolism throughout development. ATP-ABP targets include kinases, phosphatases, NAD- and FAD-dependent enzymes, RNA/DNA helicases, and others. The serine hydrolase-targeting probe detected changes in the activities of several proteases during the course of lung development, yielding insights into protein turnover at different stages of development. Select activity-based probe targets were then correlated with RNA in situ hybridization analyses of lung tissue sections.

Superphenylphosphines: Nanographene-Based Ligands That Control Coordination Geometry and Drive Supramolecular Assembly.

Tertiary phosphines remain widely utilized in synthesis, most notably as supporting ligands in metal complexes. A series of triarylphosphines bearing one to three hexa-peri-hexabenzocoronene (HBC) substituents has been prepared by an efficient divergent route. These "superphenylphosphines", P{HBC(t-Bu)5}nPh3-n (n = 1-3), form the palladium complexes PdCl2L2 and Pd2Cl4L2 where the isomer distribution in solution is dependent on the number of HBC substituents. The crystalline structures of five complexes all show intramolecular π-stacking between HBC-phosphines to form a supramolecular bidentate-like ligand that distorts the metal coordination geometry. When n = 2 or 3, the additional HBC substituents engage in intermolecular π-stacking to assemble the complexes into continuous ribbons or sheets. The phosphines adopt HBC's characteristics including strong optical absorption, green emission, and redox activity.

Dual-Readout Immunochromatographic Assay by Utilizing MnO2 Nanoflowers as the Unique Colorimetric/Chemiluminescent Probe.

Manganese dioxide nanoflowers (MnO2 NFs) were synthesized and used as a dual readout probe to develop a novel immunochromatographic test strip (ITS) for detecting pesticide residues using chlorpyrifos as the model analyte. MnO2 NFs-labeled antibody for chlorpyrifos was employed as the signal tracer for conducting the ITS. After 10 min competitive immunoreaction, the tracer antibody was captured by the immobilized immunogen in the test strip, resulting in the captured MnO2 NFs on test line. The captured MnO2 NFs led to the appearance of brown color on the test line, which could be easily observed by the naked eye as a qualitative readout. Due to the very slight colorimetric difference of chlorpyrifos at trace concentrations, the semiquantitative readout by naked eyes could not meet the demand of quantitative analysis. MnO2 NFs showed a significant effect on the luminol-H2O2 chemiluminescent (CL) system, and the CL signal driven by MnO2 NFs were used to detect the trace concentration of chlorpyrifos quantitatively. 1,3-Diphenylisobenzofuran quenching studies and TMB-H2O2 coloration assays were conducted for studying the enhancing mechanism of MnO2 NFs, which was based on the oxidant activity to decompose H2O2 for forming reactive oxygen species. Under optimal conditions, the linear range of chlorpyrifos was 0.1-50 ng/mL with a low detection limit of 0.033 ng/mL (S/N = 3). The reliability of the dual-readout ITS was successfully demonstrated by the application on traditional Chinese medicine and environmental water samples. Due to the simultaneous rapid-qualitative and sensitive-quantitative detection, the dual-readout protocol provides a promising strategy for rapid screening and field assay on various areas such as environmental monitoring and food safety.

All that is silver is not toxic: silver ion and particle kinetics reveals the role of silver ion aging and dosimetry on the toxicity of silver nanoparticles.

BACKGROUND: When suspended in cell culture medium, nano-objects composed of soluble metals such as silver can dissolve resulting in ion formation, altered particle properties (e.g. mass, morphology, etc.), and modulated cellular dose. Cultured cells are exposed not just to nanoparticles but to a complex, dynamic mixture of altered nanoparticles, unbound ions, and ion-ligand complexes. Here, three different cell types (RAW 264.7 macrophages and bone marrow derived macrophages from wild-type C57BL/6 J mice and Scavenger Receptor A deficient (SR-A(-/-)) mice) were exposed to 20 and 110 nm silver nanoparticles, and RAW 264.7 cells were exposed to freshly mixed silver ions, aged silver ions (ions incubated in cell culture medium), and ions formed from nanoparticle dissolution. The In Vitro Sedimentation, Diffusion, Dissolution, and Dosimetry Model (ISD3) was used to predict dose metrics for each exposure scenario. RESULTS: Silver nanoparticles, freshly mixed ions, and ions from nanoparticle dissolution were toxic, while aged ions were not toxic. Macrophages from SR-A(-/-) mice did not take up 20 nm silver nanoparticles as well as wild-types but demonstrated no differences in silver levels after exposure to 110 nm nanoparticles. Dose response modeling with ISD3 predicted dose metrics suggest that amount of ions in cells and area under the curve (AUC) of ion amount in cells are the most predictive of cell viability after nanoparticle and combined nanoparticle/dissolution-formed-ions exposures, respectively. CONCLUSIONS: Results of this study suggest that the unbound silver cation is the ultimate toxicant, and ions formed extracellularly drive toxicity after exposure to nanoparticles. Applying computational modeling (ISD3) to better understand dose metrics for soluble nanoparticles allows for better interpretation of in vitro hazard assessments.