Drinking water sources, quality, and associated health outcomes in Appalachian Virginia: A risk characterization study in two counties.

OBJECTIVES: In the US, violations of drinking water regulations are highest in lower-income rural areas overall, and particularly in Central Appalachia. However, data on drinking water use, quality, and associated health outcomes in rural Appalachia are limited. We sought to assess public and private drinking water sources and associated risk factors for waterborne pathogen exposures for individuals living in rural regions of Appalachian Virginia. METHODS: We administered surveys and collected tap water, bottled water, and saliva samples in lower-income households in two adjacent rural counties in southwest Virginia (bordering Kentucky and Tennessee). Water samples were tested for pH, temperature, conductivity, total coliforms, E. coli, free chlorine, nitrate, fluoride, heavy metals, and specific pathogen targets. Saliva samples were analyzed for antibody responses to potentially waterborne infections. We also shared water analysis results with households. RESULTS: We enrolled 33 households (83 individuals), 82% (n = 27) with utility-supplied water and 18% with private wells (n = 3) or springs (n = 3). 58% (n = 19) reported household incomes of <$20,000/year. Total coliforms were detected in water samples from 33% (n = 11) of homes, E. coli in 12%, all with wells or springs (n = 4), and Aeromonas, Campylobacter, and Enterobacter in 9%, all spring water (n = 3). Diarrhea was reported for 10% of individuals (n = 8), but was not associated with E. coli detection. 34% (n = 15) of saliva samples had detectable antibody responses for Cryptosporidium spp., C. jejuni, and Hepatitis E. After controlling for covariates and clustering, individuals in households with septic systems and straight pipes had significantly higher likelihoods of antibody detection (risk ratios = 3.28, 95%CI = 1.01-10.65). CONCLUSIONS: To our knowledge, this is the first study to collect and analyze drinking water samples, saliva samples, and reported health outcome data from low-income households in Central Appalachia. Our findings indicate that utility-supplied water in this region was generally safe, and individuals in low-income households without utility-supplied water or sewerage have higher exposures to waterborne pathogens.

Evaluating the utility of a high throughput thiol-containing fluorescent probe to screen for reactivity: A case study with the Tox21 library.

High-throughput screening (HTS) assays for bioactivity in the Tox21 program aim to evaluate an array of different biological targets and pathways, but a significant barrier to interpretation of these data is the lack of high-throughput screening (HTS) assays intended to identify non-specific reactive chemicals. This is an important aspect for prioritising chemicals to test in specific assays, identifying promiscuous chemicals based on their reactivity, as well as addressing hazards such as skin sensitisation which are not necessarily initiated by a receptor-mediated effect but act through a non-specific mechanism. Herein, a fluorescence-based HTS assay that allows the identification of thiol-reactive compounds was used to screen 7,872 unique chemicals in the Tox21 10K chemical library. Active chemicals were compared with profiling outcomes using structural alerts encoding electrophilic information. Random Forest classification models based on chemical fingerprints were developed to predict assay outcomes and evaluated through 10-fold stratified cross validation (CV). The mean CV Balanced Accuracy of the validation set was 0.648. The model developed shows promise as a tool to screen untested chemicals for their potential electrophilic reactivity based solely on chemical structural features.

High-throughput AR dimerization assay identifies androgen disrupting chemicals and metabolites.

Introduction: Analysis of streamlined computational models used to predict androgen disrupting chemicals revealed that assays measuring androgen receptor (AR) cofactor recruitment/dimerization were particularly indispensable to high predictivity, especially for AR antagonists. As the original dimerization assays used to develop the minimal assay models are no longer available, new assays must be established and evaluated as suitable alternatives to assess chemicals beyond the original 1,800+ supported by the current data. Here we present the AR2 assay, which is a stable, cell-based method that uses an enzyme complementation approach. Methods: Bipartite domains of the NanoLuc luciferase enzyme were fused to the human AR to quantitatively measure ligand-dependent AR homodimerization. 128 chemicals with known endocrine activity profiles including 43 AR reference chemicals were screened in agonist and antagonist modes and compared to the legacy assays. Test chemicals were rescreened in both modes using a retrofit method to incorporate robust cytochrome P450 (CYP) metabolism to assess CYP-mediated shifts in bioactivity. Results: The AR2 assay is amenable to high-throughput screening with excellent robust Z'-factors (rZ') for both agonist (0.94) and antagonist (0.85) modes. The AR2 assay successfully classified known agonists (balanced accuracy = 0.92) and antagonists (balanced accuracy = 0.79-0.88) as well as or better than the legacy assays with equal or higher estimated potencies. The subsequent reevaluation of the 128 chemicals tested in the presence of individual human CYP enzymes changed the activity calls for five compounds and shifted the estimated potencies for several others. Discussion: This study shows the AR2 assay is well suited to replace the previous AR dimerization assays in a revised computational model to predict AR bioactivity for parent chemicals and their metabolites.

E-Cigarette Liquids and Aldehyde Flavoring Agents Inhibit CYP2A6 Activity in Lung Epithelial Cells.

Certain e-liquids and aromatic aldehyde flavoring agents were previously identified as inhibitors of microsomal recombinant CYP2A6, the primary nicotine-metabolizing enzyme. However, due to their reactive nature, aldehydes may react with cellular components before reaching CYP2A6 in the endoplasmic reticulum. To determine whether e-liquid flavoring agents inhibited CYP2A6 in a cellular system, we investigated their effects on CYP2A6 using BEAS-2B cells transduced to overexpress CYP2A6. We demonstrated that two e-liquids and three aldehyde flavoring agents (cinnamaldehyde, benzaldehyde, and ethyl vanillin) exhibited dose-dependent inhibition of cellular CYP2A6.

Real-time redox adaptations in human airway epithelial cells exposed to isoprene hydroxy hydroperoxide.

While redox processes play a vital role in maintaining intracellular homeostasis by regulating critical signaling and metabolic pathways, supra-physiological or sustained oxidative stress can lead to adverse responses or cytotoxicity. Inhalation of ambient air pollutants such as particulate matter and secondary organic aerosols (SOA) induces oxidative stress in the respiratory tract through mechanisms that remain poorly understood. We investigated the effect of isoprene hydroxy hydroperoxide (ISOPOOH), an atmospheric oxidation product of vegetation-derived isoprene and a constituent of SOA, on intracellular redox homeostasis in cultured human airway epithelial cells (HAEC). We used high-resolution live cell imaging of HAEC expressing the genetically encoded ratiometric biosensors Grx1-roGFP2, iNAP1, or HyPer, to assess changes in the cytoplasmic ratio of oxidized glutathione to reduced glutathione (GSSG:GSH), and the flux of NADPH and H2O2, respectively. Non-cytotoxic exposure to ISOPOOH resulted in a dose-dependent increase of GSSG:GSH in HAEC that was markedly potentiated by prior glucose deprivation. ISOPOOH-induced increase in glutathione oxidation were accompanied by concomitant decreases in intracellular NADPH. Following ISOPOOH exposure, the introduction of glucose resulted in a rapid restoration of GSH and NADPH, while the glucose analog 2-deoxyglucose resulted in inefficient restoration of baseline GSH and NADPH. To elucidate bioenergetic adaptations involved in combatting ISOPOOH-induced oxidative stress we investigated the regulatory role of glucose-6-phosphate dehydrogenase (G6PD). A knockout of G6PD markedly impaired glucose-mediated recovery of GSSG:GSH but not NADPH. These findings reveal rapid redox adaptations involved in the cellular response to ISOPOOH and provide a live view of the dynamic regulation of redox homeostasis in human airway cells as they are exposed to environmental oxidants.

Live cell imaging of oxidative stress in human airway epithelial cells exposed to isoprene hydroxyhydroperoxide.

Exposure to respirable air particulate matter (PM2.5) in ambient air is associated with morbidity and premature deaths. A major source of PM2.5 is the photooxidation of volatile plant-produced organic compounds such as isoprene. Photochemical oxidation of isoprene leads to the formation of hydroperoxides, environmental oxidants that lead to inflammatory (IL-8) and adaptive (HMOX1) gene expression in human airway epithelial cells (HAEC). To examine the mechanism through which these oxidants alter intracellular redox balance, we used live-cell imaging to monitor the effects of isoprene hydroxyhydroperoxides (ISOPOOH) in HAEC expressing roGFP2, a sensor of the glutathione redox potential (EGSH). Non-cytotoxic exposure of HAEC to ISOPOOH resulted in a rapid and robust increase in EGSH that was independent of the generation of intracellular or extracellular hydrogen peroxide. Our results point to oxidation of GSH through the redox relay initiated by glutathione peroxidase 4, directly by ISOPOOH or indirectly by ISOPOOH-generated lipid hydroperoxides. We did not find evidence for involvement of peroxiredoxin 6. Supplementation of HAEC with polyunsaturated fatty acids enhanced ISOPOOH-induced glutathione oxidation, providing additional evidence that ISOPOOH initiates lipid peroxidation of cellular membranes. These findings demonstrate that ISOPOOH is a potent environmental airborne hydroperoxide with the potential to contribute to oxidative burden of human airway posed by inhalation of secondary organic aerosols.

Paradoxical anxiolytic effect of the 'bath salt' synthetic cathinone MDPV during early abstinence is inhibited by a chemokine CXCR4 or CCR5 receptor antagonist.

Chemokine CXCR4 and CCR5 receptors are best known as HIV co-entry receptors, but evidence that CXCR4 or CCR5 blockade reduces rewarding and locomotor-stimulant effects of psychostimulants in rats suggests a role in psychostimulant use disorders. We investigated the impact of CXCR4 or CCR5 receptor antagonism on anxiety-related effects of the synthetic cathinone 3,4-methylenedioxypyrovalerone (MDPV) in the elevated zero-maze (EZM) assay. Rats exposed to a 4-day MDPV binge dosing paradigm and tested 24 or 72 h post-treatment spent more time in the open compartment at the 24-h time point but less time at the 72-h post-binge time point. Daily administration of AMD 3100, a CXCR4 antagonist (10 mg/kg), or maraviroc, a CCR5 antagonist (2.5 mg/kg), during MDPV treatment inhibited the MDPV-induced increase in time spent in the open compartment. Neither antagonist affected the MDPV-induced reduction in time spent in the open compartment at the 72-h post-binge time point. Cocaine, administered in the same paradigm as MDPV, did not increase time spent in the open compartment 24-h post-binge, suggesting specificity to MDPV. The present results identify a surprising anxiolytic-like effect of MDPV 24 h after cessation of repeated exposure that is sensitive to chemokine CXCR4 and CCR5 receptor activity.

A Multiplex Noninvasive Salivary Antibody Assay for SARS-CoV-2 Infection and Its Application in a Population-Based Survey by Mail.

Noninvasive salivary antibody immunoassays can enable low-cost epidemiological surveillance of infections. This study involved developing and validating a multiplex suspension immunoassay on the Luminex platform to measure immunoglobulin G (IgG) responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid and spike (S) proteins, and the spike protein's S1 and S2 subunits and receptor binding domain. Multiple versions of these recombinant proteins acquired from commercial and noncommercial sources were evaluated. Assay development and validation utilized saliva and serum samples from coronavirus disease 2019 (COVID-19) cases procured from commercial sources and negative controls from a prepandemic survey. Saliva was also collected in a demonstration survey by mail involving adult individuals in the United States who were diagnosed with SARS-CoV-2 infection 15 to 80 days prior to sample collection. The survey had an 83% valid sample return rate (192 samples from 38 states). Most COVID-19 cases (93%) reported mildly symptomatic or asymptomatic infections. The final salivary assay based on the best-performing spike and nucleocapsid proteins had a sensitivity of 87.1% (95% bootstrap confidence interval, 82.1 to 91.7%) and specificity of 98.5% (95.0 to 100%) using 227 and 285 saliva samples, respectively. The same assay had 95.9% (92.8 to 98.9%) sensitivity and 100% (98.4 to 100%) specificity in serum (174 and 285 serum samples, respectively). Salivary and serum antibody responses to spike and nucleocapsid proteins were strongly correlated in 22 paired samples (r = 0.88 and r = 0.80, respectively). Antibody responses peaked at approximately 50 days postonset; greater illness severity was associated with stronger responses. This study demonstrated that a salivary antibody assay can be used in large-scale population surveys by mail to better characterize public health impacts of COVID-19. IMPORTANCE Given the enormous impacts of the COVID-19 pandemic, developing tools for population surveillance of infection is of paramount importance. This article describes the development of a multiplex immunoassay on a Luminex platform to measure salivary immunoglobulin G responses to the spike protein, its two subunits and receptor binding domain, and the nucleocapsid protein of SARS-CoV-2. The assay validation utilized serum and saliva samples from prepandemic controls and recent COVID-19 cases. A survey by mail targeting recent COVID-19 cases across the United States also demonstrated the utility of safe, at-home self-collection of saliva. By incorporating multiple SARS-CoV-2 proteins, this assay may differentiate responses to natural SARS-CoV-2 infections from responses to most vaccines. Application of this noninvasive immunoassay in COVID-19 surveillance can help provide estimates of cumulative incidence rates of symptomatic and asymptomatic infections in various communities and subpopulations, temporal patterns of antibody responses, and risk factors for infection.

CRF-5-HT interactions in the dorsal raphe nucleus and motivation for stress-induced opioid reinstatement.

RATIONALE: The serotonin (5-hydroxytryptamine, 5-HT) system plays an important role in stress-related psychiatric disorders and substance abuse. Our previous data show that stressors can inhibit 5-HT neuronal activity and release by stimulating the release of the stress neurohormone corticotropin-releasing factor (CRF) within the serotonergic dorsal raphe nucleus (DRN). The inhibitory effects of CRF on 5-HT DRN neurons are indirect, mediated by CRF-R1 receptors located on GABAergic afferents. OBJECTIVES: We tested the hypothesis that DRN CRF-R1 receptors contribute to stress-induced reinstatement of morphine-conditioned place preference (CPP). We also examined the role of this circuitry in stress-induced negative affective state with 22-kHz distress ultrasonic vocalizations (USVs), which are naturally emitted by rats in response to environmental challenges such as pain, stress, and drug withdrawal. METHODS: First, we tested if activation of CRF-R1 receptors in the DRN with the CRF-R1-preferring agonist ovine CRF (oCRF) would reinstate morphine CPP and then if blockade of CRF-R1 receptors in the DRN with the CRF-R1 antagonist NBI 35965 would attenuate swim stress-induced reinstatement of morphine CPP. Second, we tested if intra-DRN pretreatment with NBI 35965 would attenuate foot shock stress-induced 22-kHz USVs. RESULTS: Intra-DRN injection of oCRF reinstated morphine CPP, while intra-DRN injection of NBI 35965 attenuated swim stress-induced reinstatement. Moreover, intra-DRN pretreatment with NBI 35965 significantly reduced 22-kHz distress calls induced by foot shock. CONCLUSIONS: These data provide evidence that stress-induced negative affective state is mediated by DRN CRF-R1 receptors and may contribute to reinstatement of morphine CPP.

Respirometric Screening and Characterization of Mitochondrial Toxicants Within the ToxCast Phase I and II Chemical Libraries.

Mitochondrial toxicity drives several adverse health outcomes. Current high-throughput screening assays for chemically induced mitochondrial toxicity typically measure changes to mitochondrial structure and may not detect known mitochondrial toxicants. We adapted a respirometric screening assay (RSA) measuring mitochondrial function to screen ToxCast chemicals in HepG2 cells using a tiered testing strategy. Of 1042 chemicals initially screened at a singlemaximal concentration, 243 actives were identified and rescreened at 7 concentrations. Concentration-response data for 3 respiration phases confirmed activity and indicated a mechanism for 193 mitochondrial toxicants: 149 electron transport chain inhibitors (ETCi), 15 uncouplers and 29 adenosine triphosphate synthase inhibitors. Subsequently, an electron flow assay was used to identify the target complex for 84 of the 149 ETCi. Sixty reference chemicals were used to compare the RSA to existing ToxCast and Tox21 mitochondrial toxicity assays. The RSA was most predictive (accuracy = 90%) of mitochondrial toxicity. The Tox21 mitochondrial membrane potential assay was also highly predictive (accuracy = 87%) of bioactivity but underestimated the potency of well-known ETCi and provided no mechanistic information. The tiered RSA approach accurately identifies and characterizes mitochondrial toxicants acting through diverse mechanisms and at a throughput sufficient to screen large chemical inventories. The electron flow assay provides additional confirmation and detailed mechanistic understanding for ETCi, the most common type of mitochondrial toxicants among ToxCast chemicals. The mitochondrial toxicity screening approach described herein may inform hazard assessment and the in vitro bioactive concentrations used to derive relevant doses for screening level chemical assessment using new approach methodologies.

A path to early diagnosis of mild cognitive impairment and dementia: validity and reliability of the myMemCheck® self-administered screening tool.

BACKGROUND: Barriers to the early detection of mild cognitive impairment (MCI) and dementia can delay diagnosis and treatment. myMemCheck® was developed as a rapid free cognitive self-assessment tool that can be completed at home to identify older adults that would benefit from a more comprehensive cognitive evaluation. OBJECTIVE: Two prospective cross-sectional studies were conducted to examine the psychometric properties and clinical utility of myMemCheck®. METHODS: In Study 1, participants were independent living residents referred to an outpatient memory clinic (N = 59); older adults in the community and post-acute nursing home residents (N = 357) comprised Study 2. RESULTS: Psychometric analyses were performed on cognitive and psychological testing data, including myMemCheck®. myMemCheck® evidenced adequate reliability and strong construct validity. Receiver operating characteristic analysis evidenced an optional myMemCheck® cut score for identifying older adults at risk for MCI or dementia. myMemCheck® explained 25% of cognitive status beyond basic patient information. CONCLUSIONS: myMemCheck® may help fast-track the diagnostic process, facilitate appropriate referrals for cognitive and neuropsychological evaluation, reduce assessment burden in health care and prevent negative outcomes associated with undetected cognitive impairment.

Cocaine abuse and midbrain circuits: Functional anatomy of hypocretin/orexin transmission and therapeutic prospect.

Cocaine abuse remains a pervasive public health problem, and treatments thus far have proven ineffective for long-term abstinence maintenance. Intensive research on the neurobiology underlying drug abuse has led to the consideration of many candidate transmitter systems to target for intervention. Among these, the hypocretin/orexin (hcrt/ox) neuropeptide system holds largely untapped yet clinically viable therapeutic potential. Hcrt/ox originates from the hypothalamus and projects widely across the mammalian central nervous system to produce neuroexcitatory actions via two excitatory G-protein coupled receptor subtypes. Functionally, hcrt/ox promotes arousal/wakefulness and facilitates energy homeostasis. In the early 2000s, hcrt/ox transmission was shown to underlie mating behavior in male rats suggesting a novel role in reward-seeking. Soon thereafter, hcrt/ox neurons were shown to respond to drug-associated stimuli, and hcrt/ox transmission was found to facilitate motivated responding for intravenous cocaine. Notably, blocking hcrt/ox transmission using systemic or site-directed pharmacological antagonists markedly reduced motivated drug-taking as well as drug-seeking in tests of relapse. This review will unfold the current state of knowledge implicating hcrt/ox receptor transmission in the context of cocaine abuse and provide detailed background on animal models and underlying midbrain circuits. Specifically, attention will be paid to the mesoaccumbens, tegmental, habenular, pallidal and preoptic circuits. The review will conclude with discussion of recent preclinical studies assessing utility of suvorexant - the first and only FDA-approved hcrt/ox receptor antagonist - against cocaine-associated behaviors.

Indices of dentate gyrus neurogenesis are unaffected immediately after or following withdrawal from morphine self-administration compared to saline self-administering control male rats.

Opiates - including morphine - are powerful analgesics with high abuse potential. In rodents, chronic opiate exposure or self-administration negatively impacts hippocampal-dependent function, an effect perhaps due in part to the well-documented opiate-induced inhibition of dentate gyrus (DG) precursor proliferation and neurogenesis. Recently, however, intravenous (i.v.) morphine self-administration (MSA) was reported to enhance the survival of new rat DG neurons. To reconcile these disparate results, we used rat i.v. MSA to assess 1) whether a slightly-higher dose MSA paradigm also increases new DG neuron survival; 2) how MSA influences cells in different stages of DG neurogenesis, particularly maturation and survival; and 3) if MSA-induced changes in DG neurogenesis persist through a period of abstinence. To label basal levels of proliferation, rats received the S-phase marker bromodeoxyuridine (BrdU, i.p.) 24 -h prior to 21 days (D) of i.v. MSA or saline self-administration (SSA). Either immediately after SA (0-D) or after 4 weeks in the home cage (28-D withdrawal), stereology was used to quantify DG proliferating precursors (or cells in cell cycle; Ki67+ cells), neuroblast/immature neurons (DCX+ cells), and surviving DG granule cells (BrdU+ cells). Analysis revealed the number of DG cells immunopositive for these neurogenesis-relevant markers was similar between MSA and SSA rats at the 0-D or 28-D timepoints. These negative data highlight the impact experimental parameters, timepoint selection, and quantification approach have on neurogenesis results, and are discussed in the context of the large literature showing the negative impact of opiates on DG neurogenesis.

Evaluating Chemicals for Thyroid Disruption: Opportunities and Challenges with in Vitro Testing and Adverse Outcome Pathway Approaches.

BACKGROUND: Extensive clinical and experimental research documents the potential for chemical disruption of thyroid hormone (TH) signaling through multiple molecular targets. Perturbation of TH signaling can lead to abnormal brain development, cognitive impairments, and other adverse outcomes in humans and wildlife. To increase chemical safety screening efficiency and reduce vertebrate animal testing, in vitro assays that identify chemical interactions with molecular targets of the thyroid system have been developed and implemented. OBJECTIVES: We present an adverse outcome pathway (AOP) network to link data derived from in vitro assays that measure chemical interactions with thyroid molecular targets to downstream events and adverse outcomes traditionally derived from in vivo testing. We examine the role of new in vitro technologies, in the context of the AOP network, in facilitating consideration of several important regulatory and biological challenges in characterizing chemicals that exert effects through a thyroid mechanism. DISCUSSION: There is a substantial body of knowledge describing chemical effects on molecular and physiological regulation of TH signaling and associated adverse outcomes. Until recently, few alternative nonanimal assays were available to interrogate chemical effects on TH signaling. With the development of these new tools, screening large libraries of chemicals for interactions with molecular targets of the thyroid is now possible. Measuring early chemical interactions with targets in the thyroid pathway provides a means of linking adverse outcomes, which may be influenced by many biological processes, to a thyroid mechanism. However, the use of in vitro assays beyond chemical screening is complicated by continuing limits in our knowledge of TH signaling in important life stages and tissues, such as during fetal brain development. Nonetheless, the thyroid AOP network provides an ideal tool for defining causal linkages of a chemical exerting thyroid-dependent effects and identifying research needs to quantify these effects in support of regulatory decision making. https://doi.org/10.1289/EHP5297.

The Next Generation Blueprint of Computational Toxicology at the U.S. Environmental Protection Agency.

The U.S. Environmental Protection Agency (EPA) is faced with the challenge of efficiently and credibly evaluating chemical safety often with limited or no available toxicity data. The expanding number of chemicals found in commerce and the environment, coupled with time and resource requirements for traditional toxicity testing and exposure characterization, continue to underscore the need for new approaches. In 2005, EPA charted a new course to address this challenge by embracing computational toxicology (CompTox) and investing in the technologies and capabilities to push the field forward. The return on this investment has been demonstrated through results and applications across a range of human and environmental health problems, as well as initial application to regulatory decision-making within programs such as the EPA's Endocrine Disruptor Screening Program. The CompTox initiative at EPA is more than a decade old. This manuscript presents a blueprint to guide the strategic and operational direction over the next 5 years. The primary goal is to obtain broader acceptance of the CompTox approaches for application to higher tier regulatory decisions, such as chemical assessments. To achieve this goal, the blueprint expands and refines the use of high-throughput and computational modeling approaches to transform the components in chemical risk assessment, while systematically addressing key challenges that have hindered progress. In addition, the blueprint outlines additional investments in cross-cutting efforts to characterize uncertainty and variability, develop software and information technology tools, provide outreach and training, and establish scientific confidence for application to different public health and environmental regulatory decisions.

Long chain lipid hydroperoxides increase the glutathione redox potential through glutathione peroxidase 4.

BACKGROUND: Peroxidation of PUFAs by a variety of endogenous and xenobiotic electrophiles is a recognized pathophysiological process that can lead to adverse health effects. Although secondary products generated from peroxidized PUFAs have been relatively well studied, the role of primary lipid hydroperoxides in mediating early intracellular oxidative events is not well understood. METHODS: Live cell imaging was used to monitor changes in glutathione (GSH) oxidation in HAEC expressing the fluorogenic sensor roGFP during exposure to 9-hydroperoxy-10E,12Z-octadecadienoic acid (9-HpODE), a biologically important long chain lipid hydroperoxide, and its secondary product 9-hydroxy-10E,12Z-octadecadienoic acid (9-HODE). The role of hydrogen peroxide (H2O2) was examined by direct measurement and through catalase interventions. shRNA-mediated knockdown of glutathione peroxidase 4 (GPx4) was utilized to determine its involvement in the relay through which 9-HpODE initiates the oxidation of GSH. RESULTS: Exposure to 9-HpODE caused a dose-dependent increase in GSH oxidation in HAEC that was independent of intracellular or extracellular H2O2 production and was exacerbated by NADPH depletion. GPx4 was involved in the initiation of GSH oxidation in HAEC by 9-HpODE, but not that induced by exposure to H2O2 or the low molecular weight alkyl tert-butyl hydroperoxide (TBH). CONCLUSIONS: Long chain lipid hydroperoxides can directly alter cytosolic EGSH independent of secondary lipid oxidation products or H2O2 production. NADPH has a protective role against 9-HpODE induced EGSH changes. GPx4 is involved specifically in the reduction of long-chain lipid hydroperoxides, leading to GSH oxidation. SIGNIFICANCE: These results reveal a previously unrecognized consequence of lipid peroxidation, which may provide insight into disease states involving lipid peroxidation in their pathogenesis.

Synthetic cathinone MDPV enhances reward function through purinergic P2X7 receptor-dependent pathway and increases P2X7 gene expression in nucleus accumbens.

BACKGROUND AND PURPOSE: Purinergic P2X7 receptors are present on neurons, astrocytes and microglia and activated by extracellular ATP. Since P2X7 receptor activation releases endogenous substrates (e.g., pro-inflammatory cytokines, dopamine, and glutamate) that facilitate psychostimulant reward and reinforcement, we investigated the hypothesis that the synthetic cathinone 3,4-methylenedioxypyrovalerone (MDPV) produces rewarding effects that are dependent on active P2X7 receptors. METHODS: Reward function was measured in male mice using intracranial self-stimulation (ICSS). MDPV (0.1, 0.3, 0.5 mg/kg, SC) and a selective P2X7 antagonist (A438079) (5, 10, 50 mg/kg, IP) were tested alone and in combination. In separate mice, gene and protein expression of P2X7 and mitochondrial adenosine triphosphate (ATP) synthase (an enzyme that catalyzes synthesis of ATP, an endogenous ligand for P2X7 receptors) in the nucleus accumbens (NAcc) were quantified following MDPV exposure (0.1, 0.5, 5 mg/kg, SC). KEY RESULTS: MDPV (0.5 mg/kg, SC) facilitated ICSS as quantified by a significant reduction in brain reward threshold. A438079 (5, 10, 50 mg/kg, IP) did not affect ICSS by itself; however, for combined administration, A438079 (10 mg/kg, IP) inhibited facilitation of ICSS by MDPV (0.5 mg/kg, SC). At the cellular level, MDPV exposure increased gene and protein expression of P2X7 and ATP synthase in the NAcc. CONCLUSION AND IMPLICATION: We provide evidence that a psychostimulant drug produces reward enhancement that is influenced by P2X7 receptor activity and enhances P2X7 receptor expression in the brain reward circuit.

Image-guided cranial irradiation-induced ablation of dentate gyrus neurogenesis impairs extinction of recent morphine reward memories.

Dentate gyrus adult neurogenesis is implicated in the formation of hippocampal-dependent contextual associations. However, the role of adult neurogenesis during reward-based context-dependent paradigms-such as conditioned place preference (CPP)-is understudied. Therefore, we used image-guided, hippocampal-targeted X-ray irradiation (IG-IR) and morphine CPP to explore whether dentate gyrus adult neurogenesis plays a role in reward memories created in adult C57BL/6J male mice. In addition, as adult neurogenesis appears to participate to a greater extent in retrieval and extinction of recent (<48 hr posttraining) versus remote (>1 week posttraining) memories, we specifically examined the role of adult neurogenesis in reward-associated contextual memories probed at recent and remote timepoints. Six weeks post-IG-IR or Sham treatment, mice underwent morphine CPP. Using separate groups, retrieval of recent and remote reward memories was found to be similar between IG-IR and Sham treatments. Interestingly, IG-IR mice showed impaired extinction-or increased persistence-of the morphine-associated reward memory when it was probed 24-hr (recent) but not 3-weeks (remote) postconditioning relative to Sham mice. Taken together, these data show that hippocampal-directed irradiation and the associated decrease in dentate gyrus adult neurogenesis affect the persistence of recently-but not remotely-probed reward memory. These data indicate a novel role for adult neurogenesis in reward-based memories and particularly the extinction rate of these memories. Consideration of this work may lead to better understanding of extinction-based behavioral interventions for psychiatric conditions characterized by dysregulated reward processing.

Synthetic cathinone adulteration of illegal drugs.

RATIONALE: Current prevalence estimates of synthetic cathinone ("bath salt") use may be underestimates given that traditional metrics (e.g., surveys, urinalysis) often fail to capture the emergent issue of synthetic cathinone adulteration of more common illegal drugs, such as ecstasy (3,4-methylenedioxymethamphetamine). OBJECTIVES: This review examines the evolution of synthetic cathinones and prevalence of use over the past decade in the United States. We also review methods of self-report and biological testing of these compounds as well as adverse outcomes associated with adulterated drug use. RESULTS: Synthetic cathinone use emerged in the United States by 2009 with use associated with tens of thousands of poisonings. Reported poisonings and self-reported use have substantially decreased over the past five years. However, our review suggests that current estimates of use are underestimates due to underreporting stemming primarily from unknown or unintentional use of adulterated formulations of relatively popular illegal drugs, such as ecstasy. CONCLUSIONS: While intentional synthetic cathinone use has decreased in recent years, evidence suggests that prevalence of use is underestimated. Testing of drugs and/or biological specimens can improve the accuracy of synthetic cathinone use estimates. Furthermore, we advocate that researchers and clinicians should become better aware that exposure to these potent compounds (e.g., as adulterants) often occurs unknowingly or unintentionally. To improve our understanding of synthetic cathinone adulteration, research utilizing a combinatorial approach (survey and biological testing) will help more accurately estimate the prevalence and impact of this public health issue.

2015 Beers criteria medication review in assisted living facilities.

BACKGROUND AND PURPOSE: The elderly population is expected to double by 2050 with falls and hospitalizations due to adverse drug events having a major effect on health and quality of life. With the release of the revised 2015 American Geriatrics Society (AGS) Beers criteria, usage of potentially inappropriate medications (PIMs) should be studied to determine their effect on falls and hospitalizations in frail populations such as those in assisted living facilities. METHODS: This quality improvement project used a retrospective chart review on residents from a purposive sample of two assisted living facilities in Northern Virginia. Residents were aged ≥65 and lived at the facility for at least 6 months and were not enrolled in hospice and/or palliative care or living in the dementia unit. The 2015 AGS Beers criteria were used to evaluate the effect of PIMs on falls and hospitalization rates. CONCLUSIONS: This project did not find statistical significance between PIMs and falls (p = .276). A favorable, but not statistically significant trend, was noted between PIMs and hospitalizations (p = .079). IMPLICATIONS FOR PRACTICE: Understanding the effect of PIMs on falls and hospitalizations could help providers improve prescribing practices for the elderly population who are at the greatest risk for potential adverse effects from polypharmacy.