DSS-induced damage to basement membranes is repaired by matrix replacement and crosslinking.

Basement membranes are an ancient form of animal extracellular matrix. As important structural and functional components of tissues, basement membranes are subject to environmental damage and must be repaired while maintaining functions. Little is known about how basement membranes get repaired. This paucity stems from a lack of suitable in vivo models for analyzing such repair. Here, we show that dextran sodium sulfate (DSS) directly damages the gut basement membrane when fed to adult Drosophila DSS becomes incorporated into the basement membrane, promoting its expansion while decreasing its stiffness, which causes morphological changes to the underlying muscles. Remarkably, two days after withdrawal of DSS, the basement membrane is repaired by all measures of analysis. We used this new damage model to determine that repair requires collagen crosslinking and replacement of damaged components. Genetic and biochemical evidence indicates that crosslinking is required to stabilize the newly incorporated repaired Collagen IV rather than to stabilize the damaged Collagen IV. These results suggest that basement membranes are surprisingly dynamic.

Evaluation of two in silico programs for predicting mutagenicity and carcinogenicity potential for 4-methylimidazole (4-MeI) and known metabolites.

4-Methylimidazole (4-MeI) is a nitrogen-containing heterocyclic compound that is used in the manufacture of chemicals, dyes and pharmaceuticals and may be found in a variety of foods following formation during heating. The purpose of this study was to use two different in silico programs, CASE Ultra and Toxtree, to investigate potential structure-activity relationships in 4-MeI and its metabolites for mutagenicity and carcinogenicity, and combine that information with the available literature to draw conclusions regarding the strength of the predictions observed. Neither CASE Ultra nor Toxtree identified any structural alerts that were associated with mutagenic activity. Data for 4-MeI from a single study were used in the development of the CASE Ultra mouse and rat carcinogenicity models, but no additional similar structures were identified in the carcinogenicity model training set. One metabolite, 5-methylhydantoin, was predicted to be positive in the CASE Ultra carcinogenicity male and female mouse models; positive predictivity percentages of 60.9% and 73.7%, respectively. However, low structural similarity between 5-methylhydantoin and the compounds identified in the training set (<25%) decreases confidence in the positive prediction. Three metabolites were predicted to be positive in the CASE Ultra mouse micronucleus model, but again suffered from low structural similarity. Both limited structural similarity and inconsistent responses among the other clastogenicity models suggest that additional structurally similar compounds are needed to assess the predictive capacity of these alerts for biological activity of these compounds.

Exploring current read-across applications and needs among selected U.S. Federal Agencies.

Read-across is a well-established data gap-filling technique applied for regulatory purposes. In US Environmental Protection Agency's New Chemicals Program under TSCA, read-across has been used extensively for decades, however the extent of application and acceptance of read-across among U.S. federal agencies is less clear. In an effort to build read-across capacity, raise awareness of the state of the science, and work towards a harmonization of read-across approaches across U.S. agencies, a new read-across workgroup was established under the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM). This is one of several ad hoc groups ICCVAM has convened to implement the ICCVAM Strategic Roadmap. In this article, we outline the charge and scope of the workgroup and summarize the current applications, tools used, and needs of the agencies represented on the workgroup for read-across. Of the agencies surveyed, the Environmental Protection Agency had the greatest experience in using read-across whereas other agencies indicated that they would benefit from gaining a perspective of the landscape of the tools and available guidance. Two practical case studies are also described to illustrate how the read-across approaches applied by two agencies vary on account of decision context.

Data-driven process to improve VA surgical flow.

Purpose During years 2014-2016, Veterans Health Administration National Surgery Office conducted a surgical flow improvement initiative (SFII) to assist low-performing surgery programs to improve their operating room efficiency (ORE). The initiative was co-sponsored by VHA National Surgery Office and VHA Office of Systems Redesign and Improvement. The paper aims to discuss this issue. Design/methodology/approach An SFII algorithm, based on first-time-start (FTS), cancellation rate (CR), lag time (LT) and OR utilization, assigned an ORE performance Level (1-low to 4-high) to each VA Medical Center (VAMC). In total, 15 VAMCs with low-performance surgery programs participated in SFII to assess the current state of their surgical flow processes and used redesign methods to focus on improvement objectives. Findings At the end of the project, 14 VSAs, 40 RPIWs, 45 "90-day projects" and 73 Just-Do-It's were completed with 65 percent (158/243) improvement actions and 86 percent sites improving/sustaining all four ORE metrics. There was a statistically significant difference in improvement across the three stages (baseline, improvement, sustain) for FTS (45.6-68.7 percent; F=44.74; p<0.000); CR (16.1-9.5 percent; F=34.46; p<0.000); LT (63.1-36.3 percent; F=92.00; p<0.000); OR utilization (43.4-57.7 percent; F=6.92; p<0.001) and VAMC level (1.7-3.65; F=80.11; p<0.000). The majority developed "fair to excellent" sustainment (91 percent) and spread (82 percent) plans. The projected annual estimated return-on-investment was $27,949,966. Originality/value The SFII successfully leveraged a small number of faculty, coaches, and industrial engineers to produce significant improvement in ORE across a large national integrated health care network. This strategy can serve healthcare leaders in managing complex healthcare issues in their facilities.

Effect of nonheme iron-containing ferritin Dpr in the stress response and virulence of pneumococci.

Streptococcus pneumoniae (pneumococcus) produces hydrogen peroxide as a by-product of metabolism and provides a competitive advantage against cocolonizing bacteria. As pneumococci do not produce catalase or an inducible regulator of hydrogen peroxide, the mechanism of resistance to hydrogen peroxide is unclear. A gene responsible for resistance to hydrogen peroxide and iron in other streptococci is that encoding nonheme iron-containing ferritin, dpr, but previous attempts to study this gene in pneumococcus by generating a dpr mutant were unsuccessful. In the current study, we found that dpr is in an operon with the downstream genes dhfr and clpX. We generated a dpr deletion mutant which displayed normal early-log-phase and mid-log-phase growth in bacteriologic medium but survived less well at stationary phase; the addition of catalase partially rescued the growth defect. We showed that the dpr mutant is significantly more sensitive to pH, heat, iron concentration, and oxidative stress due to hydrogen peroxide. Using a mouse model of colonization, we also showed that the dpr mutant displays a reduced ability to colonize and is more rapidly cleared from the nasopharynx. Our results thus suggest that Dpr is important for pneumococcal resistance to stress and for nasopharyngeal colonization.

Toll-like receptor 2-dependent protection against pneumococcal carriage by immunization with lipidated pneumococcal proteins.

Infections with Streptococcus pneumoniae cause substantial morbidity and mortality, particularly in children in developing nations. Polysaccharide-conjugate vaccines provide protection against both invasive disease and colonization, but their use in developing countries is limited by restricted serotype coverage and expense of manufacture. Using proteomic screens, we recently identified several antigens that protected mice from pneumococcal colonization in a CD4(+) T cell- and interleukin-17A (IL-17A)-dependent manner. Since several of these proteins are lipidated, we hypothesized that their immunogenicity and impact on colonization are in part due to activation of Toll-like receptor 2 (TLR2), a receptor for lipoproteins. Here we show that lipidated versions of the antigens elicited significantly higher activation of both human embryonic kidney cells engineered to express TLR2 (HEK-TLR2) and wild-type (WT) murine macrophages than nonlipidated mutant antigens. Lipoprotein-stimulated secretion of proinflammatory cytokines was ∼10× to ∼100× lower in murine TLR2-deficient macrophages than in WT macrophages. Subcutaneous immunization of C57BL/6 mice with protein subunit vaccines containing one or two of these lipoproteins or protein fusion constructs bearing N-terminal lipid adducts elicited a robust IL-17A response and a significant reduction in colonization compared with immunization with alum alone. In contrast, immunization of Tlr2(-/-) mice elicited no detectable IL-17A response and no protection against pneumococcal colonization. These experiments suggest that the lipid moieties enhance the immunogenicity and protective efficacy of pneumococcal TH17 antigens through activation of TLR2. Thus, triggering TLR2 with an antigen-specific protein subunit formulation is a possible strategy for the development of a serotype-independent pneumococcal vaccine that would reduce pneumococcal carriage.

Development of SNAP-tag fluorogenic probes for wash-free fluorescence imaging.

The ability to specifically attach chemical probes to individual proteins represents a powerful approach to the study and manipulation of protein function in living cells. It provides a simple, robust and versatile approach to the imaging of fusion proteins in a wide range of experimental settings. However, a potential drawback of detection using chemical probes is the fluorescence background from unreacted or nonspecifically bound probes. In this report we present the design and application of novel fluorogenic probes for labeling SNAP-tag fusion proteins in living cells. SNAP-tag is an engineered variant of the human repair protein O(6)-alkylguanine-DNA alkyltransferase (hAGT) that covalently reacts with benzylguanine derivatives. Reporter groups attached to the benzyl moiety become covalently attached to the SNAP tag while the guanine acts as a leaving group. Incorporation of a quencher on the guanine group ensures that the benzylguanine probe becomes highly fluorescent only upon labeling of the SNAP-tag protein. We describe the use of intramolecularly quenched probes for wash-free labeling of cell surface-localized epidermal growth factor receptor (EGFR) fused to SNAP-tag and for direct quantification of SNAP-tagged ß-tubulin in cell lysates. In addition, we have characterized a fast-labeling variant of SNAP-tag, termed SNAP(f), which displays up to a tenfold increase in its reactivity towards benzylguanine substrates. The presented data demonstrate that the combination of SNAP(f) and the fluorogenic substrates greatly reduces the background fluorescence for labeling and imaging applications. This approach enables highly sensitive spatiotemporal investigation of protein dynamics in living cells.

Implementation context for addressing social needs in a learning health system: a qualitative study.

INTRODUCTION: Unmet social needs contribute to growing health disparities and rising health care costs. Strategies to collect and integrate information on social needs into patients' electronic health records (EHRs) show promise for connecting patients with community resources. However, gaps remain in understanding the contextual factors that impact implementing these interventions in clinical settings. METHODS: We conducted qualitative interviews with patients and focus groups with providers (January-September 2020) in two primary care clinics to inform the implementation of a module that collects and integrates patient-reported social needs information into the EHR. Questions addressed constructs within the Theoretical Framework for Acceptability and the Consolidated Framework for Implementation Research. Data were coded deductively using team-based framework analysis, followed by inductive coding and matrix analyses. RESULTS: Forty patients participated in interviews, with 20 recruited at the clinics and 20 from home. Two focus groups were conducted with a total of 12 providers. Factors salient to acceptability and feasibility included patients' discomfort answering sensitive questions, concerns about privacy, difficulty reading/understanding module content, and technological literacy. Rapport with providers was a facilitator for patients to discuss social needs. Providers stressed that limited time with patients would be a barrier, and expressed concerns about the lack of available community resources. CONCLUSION: Findings highlight the need for flexible approaches to assessing and discussing social needs with patients. Feasibility of the intervention is contingent upon support from the health system to facilitate social needs assessment and discussion. Further study of availability of community resources is needed to ensure intervention effectiveness.

Characterization of non-lipid autotaxin inhibitors.

Autotaxin (ATX) is a member of the ecto-nucleotide pyrophosphatase/phosphodiesterase (NPP) family and is a lysophospholipase D that cleaves the choline headgroup from lysophosphatidylcholine to generate the bioactive lipid lysophosphatidic acid (LPA). Enhanced expression of ATX and specific receptors for LPA in numerous cancer cell types has created an interest in studying ATX as a potential chemotherapeutic target. Likewise, ATX has been linked to several additional human diseases including multiple sclerosis, diabetes, obesity, neuropathic pain, and Alzheimer's disease. ATX inhibitors reported to date consist of metal ion chelators, lipid-like product analogs, and non-lipid small molecules. In the current research, we examined the pharmacology of the best of our previously reported non-lipid small molecule inhibitors. Here, these six inhibitors were studied utilizing the synthetic fluorescent lysophospholipid substrate FS-3, the nucleotide substrate pNP-TMP and the endogenous substrate LPC (16:0). All six compounds inhibited FS-3 hydrolysis >or=50%, whereas only three inhibited the hydrolysis of pNP-TMP to this degree. None of the six compounds blocked LPC 16:0 hydrolysis within the desired 50% inhibition range. The most potent analog (5, H2L 7905958) displayed an IC(50) of 1.6microM (K(i)=1.9microM, competitive inhibition) with respect to ATX-mediated FS-3 hydrolysis and an IC(50) of 1.2microM (K(i)=K(i)(')=6.5microM, non-competitive inhibition) against ATX-mediated pNP-TMP hydrolysis. All six inhibitors were specific for ATX as they were without affect on two additional lipid preferring NPP isoforms.

Real-Time Analysis of Individual Ebola Virus Glycoproteins Reveals Pre-Fusion, Entry-Relevant Conformational Dynamics.

The Ebola virus (EBOV) envelope glycoprotein (GP) mediates the fusion of the virion membrane with the membrane of susceptible target cells during infection. While proteolytic cleavage of GP by endosomal cathepsins and binding of the cellular receptor Niemann-Pick C1 protein (NPC1) are essential steps for virus entry, the detailed mechanisms by which these events promote membrane fusion remain unknown. Here, we applied single-molecule Förster resonance energy transfer (smFRET) imaging to investigate the structural dynamics of the EBOV GP trimeric ectodomain, and the functional transmembrane protein on the surface of pseudovirions. We show that in both contexts, pre-fusion GP is dynamic and samples multiple conformations. Removal of the glycan cap and NPC1 binding shift the conformational equilibrium, suggesting stabilization of conformations relevant to viral fusion. Furthermore, several neutralizing antibodies enrich alternative conformational states. This suggests that these antibodies neutralize EBOV by restricting access to GP conformations relevant to fusion. This work demonstrates previously unobserved dynamics of pre-fusion EBOV GP and presents a platform with heightened sensitivity to conformational changes for the study of GP function and antibody-mediated neutralization.

Developments in single-molecule and single-particle fluorescence-based approaches for studying viral envelope glycoprotein dynamics and membrane fusion.

Fusion of viral and cellular membranes is an essential step in the entry pathway of all enveloped viruses. This is a dynamic and multistep process, which has been extensively studied, resulting in the endpoints of the reaction being firmly established, and many essential cellular factors identified. What remains is to elucidate the dynamic events that underlie this process, including the order and timing of glycoprotein conformational changes, receptor-binding events, and movement of the glycoprotein on the surface of the virion. Due to the inherently asynchronous nature of these dynamics, there has been an increased focus on the study of single virions and single molecules. These techniques provide researchers the high precision and resolution necessary to bridge the gaps in our understanding of viral membrane fusion. This review highlights the advancement of single-molecule and single-particle fluorescence-based techniques, with a specific focus on how these techniques have been used to study the dynamic nature of the viral fusion pathway.

Collision-induced dissociation tandem mass spectrometry of desferrioxamine siderophore complexes from electrospray ionization of UO2(2+), Fe3+ and Ca2+ solutions.

Desferrioxamine (DEF) is a trihydroxamate siderophore typical of those produced by bacteria and fungi for the purpose of scavenging Fe(3+) from environments where the element is in short supply. Since this class of molecules has excellent chelating properties, reaction with metal contaminants such as actinide species can also occur. The complexes that are formed can be mobile in the environment. Because the natural environment is extremely diverse, strategies are needed for the identification of metal complexes in aqueous matrices having a high degree of chemical heterogeneity, and electrospray ionization mass spectrometry (ESI-MS) has been highly effective for the characterization of siderophore-metal complexes. In this study, ESI-MS of solutions containing DEF and either UO(2)(2+), Fe(3+) or Ca(2+) resulted in generation of abundant singly charged ions corresponding to [UO(2)(DEF - H)](+), [Fe(DEF - 2H)](+) and [Ca(DEF - H)](+). In addition, less abundant doubly charged ions were produced. Mass spectrometry/mass spectrometry (MS/MS) studies of collision-induced dissociation (CID) reactions of protonated DEF and metal-DEF complexes were contrasted and rationalized in terms of ligand structure. In all cases, the most abundant fragmentation reactions involved cleavage of the hydroxamate moieties, consistent with the idea that they are most actively involved with metal complexation. Singly charged complexes tended to be dominated by cleavage of a single hydroxamate, while competitive fragmentation between two hydroxamate moieties increased when the doubly charged complexes were considered. Rupture of amide bonds was also observed, but these were in general less significant than the hydroxamate fragmentations. Several lower abundance fragmentations were unique to the metal examined: abundant loss of H(2)O occurred only for the singly charged UO(2)(2+) complex. Further, NH(3) was eliminated only from the singly charged Fe(3+) complex; this and fragmentation of C-C and C-N bonds derived from neither the hydroxamate nor the amide groups suggested that Fe(3+) insertion reactions were competing with ligand complexation. In no experiments were coordinating solvent molecules observed, attached either to the intact complexes or to the fragment ions, which indicated that both intact DEF and its fragments were occupying all of the coordination sites around the metal centers. This conclusion was based on previous experiments that showed that undercoordinated UO(2)(2+) and Fe(3+) readily added H(2)O and methanol in the ESI quadrupole ion trap mass spectrometer that was used in this study.

Azide alkyne cycloaddition facilitated by hexanuclear rhenium chalcogenide cluster complexes.

Two hexanuclear rhenium clusters containing azide ligands, [Re6Se8(PEt3)5(N3)]BF4 and [Re6Se8(PEt3)4(N3)2], were synthesized from the analogous pyridine complexes and fully characterized. Studies show that [Re6Se8(PEt3)5(N3)]BF4 reacts with activated alkynes, dimethyl acetylenedicarboxylate and methyl 4-hydroxyhex-2-yneoate, to form the triazolate cluster complexes [Re6Se8(PEt3)5(L1 or L2)]BF4 (where L1 = 4,5-bis(methoxycarbonyl)-1,2,3-triazol-2-yl and L2 = 4-methoxycarbonyl-5-(1-propanol)-1,2,3-triazol-2-yl). The bis-triazolato complex, cis-[Re6Se8(PEt3)4(L1)2] was also prepared via a similar reaction starting with cis-[Re6Se8(PEt3)4(N3)2] demonstrating that these clusters can promote two azide moieties to undergo heterocyclic ring formation. The structures of [Re6Se8(PEt3)5(N3)]BF4, [Re6Se8(PEt3)4(N3)2], and [Re6Se8(PEt3)5(L1)](BF4), were determined by single-crystal X-ray diffraction analysis. In addition, studies involving the alkylation of [Re6Se8(PEt3)5(L1)]BF4 with benzyl bromide and methyl iodide are reported.

Pharmacophore development and application toward the identification of novel, small-molecule autotaxin inhibitors.

Autotaxin (ATX) is a secreted glycoprotein with lysophospholipase D (LPLD) activity that generates the bioactive lipid lysophosphatidic acid (LPA) from lysophosphatidylcholine (LPC). Both ATX and LPA have been linked to the promotion and progression of cancer as well as cardiovascular disease and obesity. Despite the fact that ATX inhibitors have the potential to be useful chemotherapeutics for multiple indications, few examples of potent ATX inhibitors are described in the current literature. Here we describe the development of pharmacophore models for the inhibition of ATX by nonlipids and apply these tools to the discovery of additional ATX inhibitors using the NCI open chemical repository database. From this database of > 250,000 compounds, 168 candidate inhibitors were identified. Of these candidates, 106 were available for testing and 33 were identified as active (those that inhibited ATX activity by > or =50% at a single 10 microM concentration), a 31% hit rate. Five of these compounds had IC(50) < 1.5 microM and the most potent compound possessed a K(i) of 271 nM.

Optimization of a pipemidic acid autotaxin inhibitor.

Autotaxin (ATX, NPP2) has recently been shown to be the lysophospholipase D responsible for synthesis of the bioactive lipid lysophosphatidic acid (LPA). LPA has a well-established role in cancer, and the production of LPA is consistent with the cancer-promoting actions of ATX. Increased ATX and LPA receptor expression have been found in numerous cancer cell types. The current study has combined ligand-based computational approaches (binary quantitative structure-activity relationship), medicinal chemistry, and experimental enzymatic assays to optimize a previously identified small molecule ATX inhibitor, H2L 7905958 (1). Seventy prospective analogs were analyzed via computational screening, from which 30 promising compounds were synthesized and screened to assess efficacy, potency, and mechanism of inhibition. This approach has identified four analogs as potent as or more potent than the lead. The most potent analog displayed an IC(50) of 900 nM with respect to ATX-mediated FS-3 hydrolysis with a K(i) of 700 nM, making this compound approximately 3-fold more potent than the previously described lead.

Chlorpyrifos and chlorpyrifos-oxon inhibit axonal growth by interfering with the morphogenic activity of acetylcholinesterase.

A primary role of acetylcholinesterase (AChE) is regulation of cholinergic neurotransmission by hydrolysis of synaptic acetylcholine. In the developing nervous system, however, AChE also functions as a morphogenic factor to promote axonal growth. This raises the question of whether organophosphorus pesticides (OPs) that are known to selectively bind to and inactivate the enzymatic function of AChE also interfere with its morphogenic function to perturb axonogenesis. To test this hypothesis, we exposed primary cultures of sensory neurons derived from embryonic rat dorsal root ganglia (DRG) to chlorpyrifos (CPF) or its oxon metabolite (CPFO). Both OPs significantly decreased axonal length at concentrations that had no effect on cell viability, protein synthesis or the enzymatic activity of AChE. Comparative analyses of the effects of CPF and CPFO on axonal growth in DRG neurons cultured from AChE nullizygous (AChE -/-) versus wild type (AChE +/+) mice indicated that while these OPs inhibited axonal growth in AChE+/+ DRG neurons, they had no effect on axonal growth in AChE -/- DRG neurons. However, transfection of AChE -/- DRG neurons with cDNA encoding full-length AChE restored the wild type response to the axon inhibitory effects of OPs. These data indicate that inhibition of axonal growth by OPs requires AChE, but the mechanism involves inhibition of the morphogenic rather than enzymatic activity of AChE. These findings suggest a novel mechanism for explaining not only the functional deficits observed in children and animals following developmental exposure to OPs, but also the increased vulnerability of the developing nervous system to OPs.

Investigation of intramolecular proton migration in a series of model, metal-cationized tripeptides using in situ generation of an isotope label.

In this study we used an isotope label, generated in situ, to investigate intramolecular proton migration or scrambling during formation of [b(2)+17+Li](+) products by collision-induced dissociation (CID) of Li(+)-cationized tripeptides. To generate the isotope label, we used a McLafferty-type rearrangement of N-terminally acetylated, C-terminal peptide tert-butyl esters in which all amide positions were exchanged with deuterium. Using a set of small, model peptides, we show that intramolecular proton scrambling occurs during CID, particularly amongst adjacent sites along a peptide backbone, on the time scales employed for low-energy collisional activation in an ion-trap mass spectrometer.

Chlorpyrifos exerts opposing effects on axonal and dendritic growth in primary neuronal cultures.

Evidence that children are widely exposed to organophosphorus pesticides (OPs) and that OPs cause developmental neurotoxicity in animal models raises significant concerns about the risks these compounds pose to the developing human nervous system. Critical to assessing this risk is identifying specific neurodevelopmental events targeted by OPs. Observations that OPs alter brain morphometry in developing rodents and inhibit neurite outgrowth in neural cell lines suggest that OPs perturb neuronal morphogenesis. However, an important question yet to be answered is whether the dysmorphogenic effect of OPs reflects perturbation of axonal or dendritic growth. We addressed this question by quantifying axonal and dendritic growth in primary cultures of embryonic rat sympathetic neurons derived from superior cervical ganglia (SCG) following in vitro exposure to chlorpyrifos (CPF) or its metabolites CPF-oxon (CPFO) and trichloropyridinol (TCP). Axon outgrowth was significantly inhibited by CPF or CPFO, but not TCP, at concentrations > or =0.001 microM or 0.001 nM, respectively. In contrast, all three compounds enhanced BMP-induced dendritic growth. Acetylcholinesterase was inhibited only by the highest concentrations of CPF (> or =1 microM) and CPFO (> or =1 nM); TCP had no effect on this parameter. In summary, these compounds perturb neuronal morphogenesis via opposing effects on axonal and dendritic growth, and both effects are independent of acetylcholinesterase inhibition. These findings have important implications for current risk assessment practices of using acetylcholinesterase inhibition as a biomarker of OP neurotoxicity and suggest that OPs may disrupt normal patterns of neuronal connectivity in the developing nervous system.

Polychlorinated biphenyls induce caspase-dependent cell death in cultured embryonic rat hippocampal but not cortical neurons via activation of the ryanodine receptor.

Perinatal exposure to polychlorinated biphenyls (PCBs) is linked to cognitive deficits in humans and experimental animals; however, the mechanism(s) underlying this effect remain speculative. Apoptosis is essential to normal brain development, and perturbation of normal spatiotemporal patterns of apoptosis can cause persistent neural deficits. We tested the hypothesis that PCBs alter apoptosis in neuronal cell types critical to cognitive function. Primary cultures of rat cortical and hippocampal neurons were treated for 48 h with Aroclor 1254 or the congeners PCB 77 and 47, which represent coplanar and noncoplanar PCBs that bind the arylhydrocarbon receptor (AhR) with high and low affinity, respectively. Using Hoechst dye and an ELISA for DNA oligonucleosomes, we observed that Aroclor 1254 (10 microM) and PCB 47 (1 microM) significantly increased DNA fragmentation in hippocampal but not cortical neurons, and this effect was blocked by the caspase inhibitors, z-VAD-fmk and DEVD-CHO. In contrast, PCB 77 had no effect on apoptosis in either neuronal cell type, suggesting that PCB-induced apoptosis occurs independent of the AhR. The proapoptotic activity of PCBs was inhibited by the ryanodine receptor (RyR) antagonist FLA 365 and by the antioxidant alpha-tocopherol but not by antagonists of the IP(3) receptor (xestospongin C), L-type calcium channel (verapamil), or NMDA receptor (APV). These data indicate that noncoplanar PCBs induce apoptosis in hippocampal neurons subsequent to RyR activation and increased reactive oxygen species and suggest that altered regional profiles of apoptosis may be an important mechanism underlying the developmental neurotoxicity of PCBs.

Mechanisms of organophosphate insecticide-induced airway hyperreactivity.

It has been suggested that pesticide exposure may be a contributing factor underlying the increased incidence of asthma in the United States and other industrialized nations. To test this hypothesis, airway hyperreactivity was measured in guinea pigs exposed to chlorpyrifos, a widely used organophosphate pesticide. Electrical stimulation of the vagus nerves caused frequency-dependent bronchoconstriction that was significantly potentiated in animals 24 h or 7 days after a single subcutaneous injection of either 390 mg/kg or 70 mg/kg of chlorpyrifos, respectively. Mechanisms by which chlorpyrifos may cause airway hyperreactivity include inhibition of acetylcholinesterase (AChE) or dysfunction of M3 muscarinic receptors on airway smooth muscle or of autoinhibitory M2 muscarinic receptors on parasympathetic nerves in the lung. AChE activity in the lung was significantly inhibited 24 h after treatment with 390 mg/kg of chlorpyrifos, but not 7 days after injection of 70 mg/kg of chlorpyrifos. Acute exposure to eserine (250 microg/ml) also significantly inhibited lung AChE but did not potentiate vagally induced bronchoconstriction. Neuronal M2 receptor function was tested using the M2 agonist pilocarpine, which inhibits vagally induced bronchoconstriction in control animals. In chlorpyrifos-treated animals, pilocarpine dose-response curves were shifted significantly to the right, demonstrating decreased responsiveness of neuronal M2 receptors. In contrast, chlorpyrifos treatment did not alter methacholine-induced bronchoconstriction, suggesting that chlorpyrifos does not alter M3 muscarinic receptor function on airway smooth muscle. These data demonstrate that organophosphate insecticides can cause airway hyperreactivity in the absence of AChE inhibition by decreasing neuronal M2 receptor function.