Biotransformation of Efavirenz and Proteomic Analysis of Cytochrome P450s and UDP-Glucuronosyltransferases in Mouse, Macaque, and Human Brain-Derived In Vitro Systems.

Antiretroviral drugs such as efavirenz (EFV) are essential to combat human immunodeficiency virus (HIV) infection in the brain, but little is known about how these drugs are metabolized locally. In this study, the cytochrome P450 (P450) and UDP-glucuronosyltransferase (UGT)-dependent metabolism of EFV was probed in brain microsomes from mice, cynomolgus macaques, and humans as well as primary neural cells from C57BL/6N mice. Utilizing ultra high performance liquid chromatography high-resolution mass spectrometry (uHPLC-HRMS), the formation of 8-hydroxyefavirenz (8-OHEFV) from EFV and the glucuronidation of P450-dependent metabolites 8-OHEFV and 8,14-dihydroxyefavirenz (8,14-diOHEFV) were observed in brain microsomes from all three species. The direct glucuronidation of EFV, however, was only detected in cynomolgus macaque brain microsomes. In primary neural cells treated with EFV, microglia were the only cell type to exhibit metabolism, forming 8-OHEFV only. In cells treated with the P450-dependent metabolites of EFV, glucuronidation was detected only in cortical neurons and astrocytes, revealing that certain aspects of EFV metabolism are cell type specific. Untargeted and targeted proteomics experiments were used to identify the P450s and UGTs present in brain microsomes. Eleven P450s and 11 UGTs were detected in human brain microsomes, whereas seven P450s and 14 UGTs were identified in mouse brain microsomes and 15 P450s and four UGTs, respectively, were observed in macaque brain microsomes. This was the first time many of these enzymes have been noted in brain microsomes at the protein level. This study indicates the potential for brain metabolism to contribute to pharmacological and toxicological outcomes of EFV in the brain. SIGNIFICANCE STATEMENT: Metabolism in the brain is understudied, and the persistence of human immunodeficiency virus (HIV) infection in the brain warrants the evaluation of how antiretroviral drugs such as efavirenz are metabolized in the brain. Using brain microsomes, the metabolism of efavirenz by both cytochrome P450s (P450s) and UDP-glucuronosyltransferases (UGTs) is established. Additionally, proteomics of brain microsomes characterizes P450s and UGTs in the brain, many of which have not yet been noted in the literature at the protein level.

Achieving a Deeper Understanding of Drug Metabolism and Responses Using Single-Cell Technologies.

Recent advancements in single-cell technologies have enabled detection of RNA, proteins, metabolites, and xenobiotics in individual cells, and the application of these technologies has the potential to transform pharmacological research. Single-cell data has already resulted in the development of human and model species cell atlases, identifying different cell types within a tissue, further facilitating the characterization of tumor heterogeneity, and providing insight into treatment resistance. Research discussed in this review demonstrates that distinct cell populations express drug metabolizing enzymes to different extents, indicating there may be variability in drug metabolism not only between organs, but within tissue types. Additionally, we put forth the concept that single-cell analyses can be used to expose underlying variability in cellular response to drugs, providing a unique examination of drug efficacy, toxicity, and metabolism. We will outline several of these techniques: single-cell RNA-sequencing and mass cytometry to characterize and distinguish different cell types, single-cell proteomics to quantify drug metabolizing enzymes and characterize cellular responses to drug, capillary electrophoresis-ultrasensitive laser-induced fluorescence detection and single-probe single-cell mass spectrometry for detection of drugs, and others. Emerging single-cell technologies such as these can comprehensively characterize heterogeneity in both cell-type-specific drug metabolism and response to treatment, enhancing progress toward personalized and precision medicine. SIGNIFICANCE STATEMENT: Recent technological advances have enabled the analysis of gene expression and protein levels in single cells. These types of analyses are important to investigating mechanisms that cannot be elucidated on a bulk level, primarily due to the variability of cell populations within biological systems. Here, we summarize cell-type-specific drug metabolism and how pharmacologists can utilize single-cell approaches to obtain a comprehensive understanding of drug metabolism and cellular heterogeneity in response to drugs.

Assessment of particulate matter toxicity and physicochemistry at the Claim 28 uranium mine site in Blue Gap, AZ.

Thousands of abandoned uranium mines (AUMs) exist in the western United States. Due to improper remediation, windblown dusts generated from AUMs are of significant community concern. A mobile inhalation lab was sited near an AUM of high community concern ("Claim 28") with three primary objectives: to (1) determine the composition of the regional ambient particulate matter (PM), (2) assess meteorological characteristics (wind speed and direction), and (3) assess immunological and physiological responses of mice after exposures to concentrated ambient PM (or CAPs). C57BL/6 and apolipoprotein E-null (ApoE-/-) mice were exposed to CAPs in AirCARE1 located approximately 1 km to the SW of Claim 28, for 1 or 28 days for 4 hr/day at approximately 80 µg/m3 CAPs. Bronchoalveolar lavage fluid (BALF) analysis revealed a significant influx of neutrophils after a single-day exposure in C57BL/6 mice (average PM2.5 concentration = 68 µg/m3). Lungs from mice exposed for 1 day exhibited modest increases in Tnfa and Tgfb mRNA levels in the CAPs exposure group compared to filtered air (FA). Lungs from mice exposed for 28 days exhibited reduced Tgfb (C57BL/6) and Tnfa (ApoE-/-) mRNA levels. Wind direction was typically moving from SW to NE (away from the community) and, while detectable in all samples, uranium concentrations in the PM2.5 fraction were not markedly different from published-reported values. Overall, exposure to CAPs in the region of the Blue GAP Tachee's Claim-28 uranium mine demonstrated little evidence of overt pulmonary injury or inflammation or ambient air contamination attributed to uranium or vanadium.

Development of prehospital assessment findings associated with massive transfusion.

BACKGROUND: Massive transfusion is frequently a component of the resuscitation of combat casualties. Because blood supplies may be limited, activation of a walking blood bank and mobilization of necessary resources must occur in a timely fashion. The development of a risk prediction model to guide clinicians for early transfusion in the prehospital setting was sought. STUDY DESIGN AND METHODS: This is a secondary analysis of a previously described data set from the Department of Defense Trauma Registry from January 2007 to August 2016 focusing on casualties undergoing massive transfusion. Serious injury was defined based on an Abbreviated Injury Scale score of 3 or greater by body region. The authors constructed multiple imputations of the model for risk prediction development. Efforts were made to internally validate the model. RESULTS: Within the data set, there were 15540 patients, of which 1238 (7.9%) underwent massive transfusion. In the body region injury scale model, explosive injuries (odds ratio [OR], 3.78), serious extremity injuries (OR, 6.59), and tachycardia >120/min (OR, 5.61) were most strongly associated with receiving a massive transfusion. In the simplified model, major amputations (OR, 17.02), tourniquet application (OR, 6.66), and tachycardia >120 beats/min (OR, 8.72) were associated with massive transfusion. Both models had area under the curve receiver operating characteristic values of greater than 0.9 for the model and bootstrap forest analysis. CONCLUSION: In the body region injury scale model, explosive mechanisms, serious extremity injuries, and tachycardia were most strongly associated with massive transfusion. In the simplified model, major amputations, tourniquet application, and tachycardia were most strongly associated.

An analysis of the pediatric casualties undergoing massive transfusion in Iraq and Afghanistan.

BACKGROUND: Existing data on pediatric massive transfusion as part of trauma resuscitation is limited. We report the characteristics of pediatric casualties associated with undergoing massive transfusion at US military treatment facilities during combat operations in Iraq and Afghanistan. METHODS: We queried the Department of Defense Trauma Registry (DODTR) for all pediatric subjects admitted to US and Coalition fixed-facility hospitals in Iraq and Afghanistan from January 2007 to January 2016. We stratified subjects by Centers for Disease Control age groupings: <1, 1-4, 5-9, 10-14, and 15-17 years. We defined a massive transfusion as 40 mL/kg of total blood products or more. RESULTS: From January 2007 through January 2016 there were 3439 pediatric casualties within the registry, of which 543 (15.7%) met criteria for receiving a massive transfusion. The median age of children undergoing massive transfusion was 9 years (IQR 5-12), male (73.4%), injured in Afghanistan (69.9%) and injured by explosives (60.4%). Compared to other pediatric casualties, subjects undergoing massive transfusion had higher composite injury severity scores (median 17 versus 9), higher incidence of tachycardia (86.8% versus 70.9%), increased incidence of hypotension (31.2% versus 7.5%), and decreased survival to hospital discharge (82.6% versus 91.6%). Specific to body regions, casualties undergoing massive transfusion more frequently had serious injuries to the head/neck (30.0% versus 22.8%), the thorax (22.8% versus 9.9%), abdomen (26.8% versus 6.9%), the extremities (42.1% versus 14.6%), while less frequently had serious injuries to the skin (5.3% versus 8.4%). All findings were significant. CONCLUSIONS: Further research is needed to better translate the lessons learned from pediatric trauma care in the combat setting into the civilian setting in developed countries. LEVEL OF EVIDENCE: 3.

Placement of Antibiotic Powder in Open Fracture Wounds during the Emergency Room (POWDER): Design and Rationale for an Investigation of the Acute Application of Topical Antibiotic Powder in Open Fracture Wounds for Infection Prophylaxis.

BACKGROUND: Open fractures are at high risk for complications both in the military and civilian setting. Treatments to prevent fractures are limited in the Role 1 (prehospital, battalion aid station) setting. The goal of this study is to assess the efficacy of topical vancomycin powder, administered within 24 hours of an open fracture injury, in the prevention of infection and infection-related complications. METHODS: The POWDER study is a multicenter, prospective, randomized controlled clinical trial using a pragmatic open-label design. We will recruit 200 long bone open fracture patients from University Hospital at University of Texas Health at San Antonio (UTHSA) and the Brooke Army Medical Center (BAMC). We will screen and randomize patients in a 1:1 ratio to receive either usual care plus 2g topical vancomycin or usual care only. The primary objective of this study is to compare the proportion of infection and infection-related complications which occur in the 2 arms. An additional objective is to develop a risk-prediction model for open fracture wound complications. CONCLUSIONS: The infection rates seen in open fractures remain alarmingly high in both combat and civilian settings. Several orthopedic surgery studies suggest vancomycin powder is effective in reducing surgical site infections when applied topically at the time of wound closure. We expect to see a reduction in infections in open fracture injuries treated acutely with vancomycin powder. This study may provide important information regarding the use of local vancomycin powder during the acute treatment of open fractures. If shown to be efficacious, vancomycin powder could provide a simple, time- and cost-effective infection prophylaxis strategy for these injuries.

Inhibition of Catechol-O-methyltransferase Does Not Alter Effort-Related Choice Behavior in a Fixed Ratio/Concurrent Chow Task in Male Mice.

Effort-related choice (ERC) tasks allow animals to choose between high-value reinforcers that require high effort to obtain and low-value/low-effort reinforcers. Dopaminergic neuromodulation regulates ERC behavior. The enzyme catechol-O-methyltransferase (COMT) metabolizes synaptically-released dopamine. COMT is the predominant regulator of dopamine turnover in regions of the brain with low levels of dopamine transporters (DATs), including the prefrontal cortex (PFC). Here, we evaluated the effects of the COMT inhibitor tolcapone on ERC performance in a touchscreen-based fixed-ratio/concurrent chow task in male mice. In this task, mice were given the choice between engaging in a fixed number of instrumental responses to acquire a strawberry milk reward and consuming standard lab chow concurrently available on the chamber floor. We found no significant effects of tolcapone treatment on either strawberry milk earned or chow consumed compared to vehicle treatment. In contrast, we found that haloperidol decreased instrumental responding for strawberry milk and increased chow consumption as seen in previously published studies. These data suggest that COMT inhibition does not significantly affect effort-related decision making in a fixed-ratio/concurrent chow task in male mice.

Toxic Effects of Particulate Matter Derived from Dust Samples Near the Dzhidinski Ore Processing Mill, Eastern Siberia, Russia.

Ambient particulate matter (PM) is associated with increased mortality and morbidity, an effect influenced by the metal components of the PM. We characterized five sediment samples obtained near a tungsten-molybdenum ore-processing complex in Zakamensk, Russia for elemental composition and PM toxicity with regard to pulmonary, vascular, and neurological outcomes. Elemental and trace metals analysis of complete sediment and PM10 (the respirable fraction, < 10 µm mass mean aerodynamic diameter) were performed using inductively coupled plasma optical emission spectrometry (ICP-OES) and mass spectrometry (ICP-MS). Sediment samples and PM10 consisted largely of silicon and iron and silicon and sodium, respectively. Trace metals including manganese and uranium in the complete sediment, as well as copper and lead in the PM10 were observed. Notably, metal concentrations were approximately 10 × higher in the PM10 than in the sediment. Exposure to 100 µg of PM10 via oropharyngeal aspiration in C56BL/6 mice resulted in pulmonary inflammation across all groups. In addition, mice exposed to three of the five PM10 samples exhibited impaired endothelial-dependent relaxation, and correlative analysis revealed associations between pulmonary inflammation and levels of lead and cadmium. A tendency for elevated cortical ccl2 and Tnf-α mRNA expression was induced by all samples and significant upregulation was noted following exposure to PM10 samples Z3 and Z4, respectively. Cortical Nqo1 mRNA levels were significantly upregulated in mice exposed to PM10 Z2. In conclusion, pulmonary exposure to PM samples from the Zakamensk region sediments induced varied pulmonary and systemic effects that may be influenced by elemental PM composition. Further investigation is needed to pinpoint putative drivers of neurological outcomes.

Respirable Uranyl-Vanadate-Containing Particulate Matter Derived From a Legacy Uranium Mine Site Exhibits Potentiated Cardiopulmonary Toxicity.

Exposure to windblown particulate matter (PM) arising from legacy uranium (U) mine sites in the Navajo Nation may pose a human health hazard due to their potentially high metal content, including U and vanadium (V). To assess the toxic impact of PM derived from Claim 28 (a priority U mine) compared with background PM, and consider the putative role of metal species U and V. Two representative sediment samples from Navajo Nation sites (Background PM and Claim 28 PM) were obtained, characterized in terms of chemistry and morphology, and fractioned to the respirable (≤ 10 µm) fraction. Mice were dosed with either PM sample, uranyl acetate, or vanadyl sulfate via aspiration (100 µg), with assessments of pulmonary and vascular toxicity 24 h later. Particulate matter samples were also examined for in vitro effects on cytotoxicity, oxidative stress, phagocytosis, and inflammasome induction. Claim 28 PM10 was highly enriched with U and V and exhibited a unique nanoparticle ultrastructure compared with background PM10. Claim 28 PM10 exhibited enhanced pulmonary and vascular toxicity relative to background PM10. Both U and V exhibited complementary pulmonary inflammatory potential, with U driving a classical inflammatory cytokine profile (elevated interleukin [IL]-1ß, tumor necrosis factor-α, and keratinocyte chemoattractant/human growth-regulated oncogene) while V preferentially induced a different cytokine pattern (elevated IL-5, IL-6, and IL-10). Claim 28 PM10 was more potent than background PM10 in terms of in vitro cytotoxicity, impairment of phagocytosis, and oxidative stress responses. Resuspended PM10 derived from U mine waste exhibit greater cardiopulmonary toxicity than background dusts. Rigorous exposure assessment is needed to gauge the regional health risks imparted by these unremediated sites.