Prevalence of fentanyl in the pediatric postmortem population from 2019 to 2023.

For the past decade, illicitly manufactured fentanyl has been a primary contributor in drug overdose deaths regardless of age. The pediatric population is particularly vulnerable to fentanyl exposure, yet there are limited case reports involving this population. Postmortem cases from 2019 to 2023 were retrospectively analyzed to determine the prevalence of fentanyl in decedents between 0 and 12 years of age. Over this time frame, the fentanyl positivity rate increased from 2.6 to 6.2% (n = 632). The most commonly reported age group was 0-4 years, with a peak around 1 year of age for toddlers. Fentanyl concentrations in blood (n = 573) ranged from 0.19 to 360 ng/mL (mean 18 ng/mL, median 6.9 ng/mL). Polydrug use was present in 428 cases; midazolam (n = 96) and methamphetamine (n = 66) were the most common drugs found concurrently in blood with fentanyl, followed by markers of illicitly manufactured fentanyl, such as xylazine (n = 23), para-fluorofentanyl (n = 18), and acetyl fentanyl (n = 17). This report contrasts the differences in postmortem pediatric fentanyl toxicology results for three groups of case histories: likely medical intervention (n = 113), pregnancy/birth related (n = 136), and inadvertent/intentional exposure (n = 196). Overall, this study provides a retrospective review of postmortem pediatric fentanyl concentrations in a variety of biological matrices and highlights the need for comprehensive toxicology testing in postmortem pediatric casework.

Drug Trends in the Teenage Postmortem Population From 2017 to 2021.

ABSTRACT: The teenage population is highly vulnerable to drug exposure, use, and misuse due to the physical and emotional development that occurs at those ages. Social influences, like the isolation experienced during the COVID-19 pandemic and social media, can exacerbate this vulnerability. To better understand the potential impact of these influences on teenage drug use, postmortem results reported by a large reference laboratory from 2017 to 2021 corresponding to the teenage population were evaluated for various drugs of misuse. These data revealed a marked increase (385%) in reported fentanyl cases and a 13% increase in positivity rate. Methamphetamine- and cocaine-positive cases also increased 126% and 54%, with a net percent positivity of +0.6% and -0.5%, respectively. Conversely, heroin showed a consistent decline in reported cases (67%) and a net decrease of 1.0% in positivity rate. In addition to commonly misused drugs, trends for other substances that are prevalent in social media and therefore may disproportionally impact teens, MDMA/MDA, mitragynine, and diphenhydramine, were also assessed. A discussion of drug-related social media trends is presented to provide additional context for the data and trends reported herein, ultimately creating a framework through which social influences on teenage drug use can be better understood.

Distribution of donepezil in postmortem casework.

Donepezil is one of the primary treatments options for patients suffering from Alzheimer's Disease. In a review of more than 2200 postmortem donepezil positive blood specimens, 76% of concentrations were higher than the proposed therapeutic range. Means and medians were similar between central blood specimens and peripheral specimens, indicating minimal postmortem redistribution. Postmortem concentrations may not reflect those circulating antemortem. Mean and median postmortem blood concentrations were approximately 3-fold higher than those in antemortem blood specimens. Additionally, in cases where antemortem blood was available for testing, large increases in donepezil concentrations were reported between antemortem and postmortem specimens without documented administration by medical personnel. Elevated blood donepezil concentrations have been reported in multiple postmortem cases where cause of death was unrelated. The blood concentrations reported in cases where donepezil did not contribute to death overlapped with those in suspected drug overdose cases where other drugs may have been present. In 4 out of 5 suspected donepezil overdose cases, blood concentrations greater than 1000 ng/mL were reported, whereas less than 1% of all postmortem blood samples reviewed achieved these concentrations. Blood concentrations greater than 1000 ng/mL should be considered contributory when a drug overdose is suspected. Postmortem donepezil concentrations should be interpreted with caution in the context of a comprehensive case history.

Chemical characterization of microplastic particles formed in airborne waste discharged from sewer pipe repairs.

Microplastic particles are of increasing environmental concern due to the widespread uncontrolled degradation of various commercial products made of plastic and their associated waste disposal. Recently, common technology used to repair sewer pipes was reported as one of the emission sources of airborne microplastics in urban areas. This research presents results of the multi-modal comprehensive chemical characterization of the microplastic particles related to waste discharged in the pipe repair process and compares particle composition with the components of uncured resin and cured plastic composite used in the process. Analysis of these materials employs complementary use of surface-enhanced Raman spectroscopy, scanning transmission X-ray spectro-microscopy, single particle mass spectrometry, and direct analysis in real-time high-resolution mass spectrometry. It is shown that the composition of the relatively large (100 µm) microplastic particles resembles components of plastic material used in the process. In contrast, the composition of the smaller (micrometer and sub-micrometer) particles is significantly different, suggesting their formation from unintended polymerization of water-soluble components occurring in drying droplets of the air-discharged waste. In addition, resin material type influences the composition of released microplastic particles. Results are further discussed to guide the detection and advanced characterization of airborne microplastics in future field and laboratory studies pertaining to sewer pipe repair technology.

Diversity of organic components in airborne waste discharged from sewer pipe repairs.

Air-discharged waste from commonly used trenchless technologies of sewer pipe repairs is an emerging and poorly characterized source of urban pollution. This study reports on the molecular-level characterization of the atmospherically discharged aqueous-phase waste condensate samples collected at four field sites of the sewer pipe repairs. The molecular composition of organic species in these samples was investigated using reversed-phase liquid chromatography coupled with a photodiode array detector and a high-resolution mass spectrometer equipped with interchangeable atmospheric pressure photoionization and electrospray ionization sources. The waste condensate components comprise a complex mixture of organic species that can partition between gas-, aqueous-, and solid-phases when water evaporates from the air-discharged waste. Identified organic species have broad variability in molecular weight, molecular structures, and carbon oxidation state, which also varied between the waste samples. All condensates contained complex mixtures of oxidized organics, N- and S-containing organics, condensed aromatics, and their functionalized derivatives that are directly released to the atmospheric environment during installations. Furthermore, semi-volatile, low volatility, and extremely low volatility organic compounds comprise 75-85% of the total compounds identified in the waste condensates. Estimates of the component-specific viscosities suggest that upon evaporation of water waste material would form the semi-solid and solid phases. The low volatilities and high viscosities of chemical components in these waste condensates will contribute to the formation of atmospheric secondary organic aerosols and atmospheric solid nanoplastic particles. Lastly, selected components expected in the condensates were quantified and found to be present at high concentrations (1-20 mg L-1) that may exceed regulatory limits.

'It just stops me from living': A qualitative study of losses experienced by women with self-reported endometriosis.

AIMS: To understand the experience of loss in Australian women with endometriosis. DESIGN: A total of 532 participants completed an online survey containing three open-ended questions relating to pelvic pain and activity loss due to endometriosis. Participants were Australian women aged between 18 and 50 years (M = 30.8, SD = 7.1) with a self-reported diagnosis of endometriosis. An inductive, qualitative approach, with template analysis was used to identify and organize themes. A pragmatic feminist perspective was used to interpret the findings. RESULTS: Three main themes were identified: the loss of liberty: 'I'm trapped in the house'; the loss of bodily autonomy: 'I can barely move/breathe/talk' and loss of connection: 'It stops me from being social'. Pain emerged as the greatest concern for participants, preventing them from the physical functioning required to participate in many of life's activities. CONCLUSIONS: The losses women with endometriosis experience are wide-reaching, restricting control and choice across multiple life domains. Losses were often unacknowledged by loved ones and healthcare providers, further impacting the physical, emotional and mental health of participants. PATIENT OR PUBLIC CONTRIBUTION: People with endometriosis were involved in the design of the study, including identifying topics of interest.

Student Confidence in Providing Healthcare to Adults With Intellectual Disability: Implications for Health Profession Curricula.

Self-perceived confidence of health professions students at one university in caring for adults with intellectual disability (ID) was examined via an electronic survey using the Therapy Confidence Scale - Intellectual Disabilities (TCS-ID). A stepwise multiple regression of data collected from 232 completed surveys revealed that prior training and prior experience were predictors of TCS-ID total score. Adults with ID experience healthcare disparities due, in part, to poor provider communication and a lack of confidence. Results from this novel study suggest that opportunities for experiential learning and training with people with ID are important considerations for health professions curricula. Further research is needed for generalizability of results.

Atmospheric emission of nanoplastics from sewer pipe repairs.

Nanoplastic particles are inadequately characterized environmental pollutants that have adverse effects on aquatic and atmospheric systems, causing detrimental effects to human health through inhalation, ingestion and skin penetration1-3. At present, it is explicitly assumed that environmental nanoplastics (EnvNPs) are weathering fragments of microplastic or larger plastic debris that have been discharged into terrestrial and aquatic environments, while atmospheric EnvNPs are attributed solely to aerosolization by wind and other mechanical forces. However, the sources and emissions of unintended EnvNPs are poorly understood and are therefore largely unaccounted for in various risk assessments4. Here we show that large quantities of EnvNPs may be directly emitted into the atmosphere as steam-laden waste components discharged from a technology commonly used to repair sewer pipes in urban areas. A comprehensive chemical analysis of the discharged waste condensate has revealed the abundant presence of insoluble colloids, which after drying form solid organic particles with a composition and viscosity consistent with EnvNPs. We suggest that airborne emissions of EnvNPs from these globally used sewer repair practices may be prevalent in highly populated urban areas5, and may have important implications for air quality and toxicological levels that need to be mitigated.

Solvent-Mediated Charge Transfer Dynamics of a Model Brown Carbon Aerosol Chromophore: Photophysics of 1-Phenylpyrrole Induced by Water Solvation.

Nitrogen heterocycles are known to be important light-absorbing chromophores in a newly discovered class of aerosols, commonly referred to as "brown carbon" (BrC) aerosols. Due to their significant absorption and spectral overlap with the solar actinic flux, these BrC chromophores steer the physical and optical properties of aerosols. To model the local aqueous solvation environment surrounding BrC chromophores, we generated cold molecular complexes with water and a prototypical BrC chromophore, 1-phenylpyrrole (1PhPy), using supersonic jet-cooling and explored their intermolecular interactions using single-conformation spectroscopy. Herein, we utilized resonant two-photon ionization (R2PI) and UV holeburning (UV HB) double-resonance spectroscopies to obtain a molecular-level understanding of the role of water microsolvation in charge transfer upon photoexcitation of 1PhPy. Quantum chemical calculations and one-dimensional discrete variable representation simulations revealed insights into the charge transfer efficacy of 1PhPy with and without addition of a single water molecule. Taken together, our results indicate that the intermolecular interactions with water guide the geometry of 1PhPy to adopt a more twisted intramolecular charge transfer (TICT) configuration, thus facilitating charge transfer from the pyrrole donor to the phenyl ring acceptor. Furthermore, the water network surrounding 1PhPy reports on the charge transfer such that the H2O solvent primarily interacts with the pyrrole ring donor in the ground state, whereas it preferentially interacts with the phenyl ring acceptor in the excited state. Large Franck-Condon activity is evident in the 1PhPy + 1H2O excitation spectrum for the water-migration vibronic bands, supporting H2O solvent reorganization upon excitation of the 1PhPy chromophore. Fluorescence measurements with increasing H2O % volume corroborated our gas-phase studies by indicating that a polar water solvation environment stabilizes the TICT configuration of 1PhPy in the excited electronic state, from which emission is observed at a lower energy compared to the locally excited configuration.

Two Fatal Intoxications Involving 3-Methoxyphencyclidine.

3- and 4-methoxyphencyclidine (3-MeO-PCP, 4-MeO-PCP), structural analogs of phencyclidine (PCP), were among the first legal PCP alternatives to show up on the novel psychoactive substances (NPS) market in Europe in the 2000s. Their structural similarities to PCP and ketamine likely contribute to their demonstrated dissociative anesthetic effects. Limited information exists in the literature about toxic and lethal concentrations of these drugs in biological samples. This case report presents the first two death cases in Washington State in which 3-MeO-PCP was identified. Alkaline drug screen analysis by gas chromatography-mass spectrometry (GC-MS) revealed a peak with a retention time similar to PCP and base peak of m/z 230. Certified reference materials for 3-and 4-MeO-PCP were obtained and the isomers were able to be distinguished based on different retention times and mass spectra. A quantitative GC-MS method was developed and validated for casework, utilizing a dynamic range of 10-1,000 ng/mL and a limit of detection of 1 ng/mL. Postmortem (peripheral/central) blood samples were analyzed using this method and the resulting concentrations were 0.63 and 3.2 mg/L of 3-MeO-PCP. Methamphetamine (0.11 mg/L) was additionally detected in the blood of one of the decedents; while the second decedent was additionally positive for ethanol (0.047 g/100 mL), bupropion (1.8 mg/L), delorazepam, paroxetine and mitragynine. The results presented in this case report are higher than previously reported concentrations in fatal cases, but the presence of polysubstance abuse is consistent with previously reported NPS intoxications. Both of these individuals were in drug rehabilitation facilities prior to their deaths; however, users continue to be drawn to 3-MeO-PCP due to its dissociative effects and its accessibility on the internet.

Concentrations of AB-CHMINACA and AB-PINACA and Driving Behavior in Suspected Impaired Driving Cases.

This article reviews case reports for 58 suspected impaired driving cases that were positive for the synthetic cannabinoids AB-CHMINACA or AB-PINACA. All cases were submitted to the Washington State Patrol Toxicology Laboratory in 2014 from either Washington State or State of Alaska law enforcement agencies. The population of drivers was predominantly male (95%), with a mean age of 28 years (range, 18-61 years). The range of blood concentrations was 0.6->10 ng/mL for AB-CHMINACA (N = 33) and 0.6-41.3 ng/mL for AB-PINACA (N = 25). Drug Recognition Expert exams were performed in 10 cases for each AB-CHMINACA and AB-PINACA. Horizontal gaze nystagmus was observed in 50 and 60% of the cases, respectively. Overall, several physiological indicators varied from those typically observed with marijuana use. The majority of these cases had very poor driving; subjects were involved in an accident, found passed out in a vehicle or were called in as a suspected impaired driver. Slurred speech, confusion, lack of coordination/dexterity and lethargy were commonly observed.

A Case Review of the First Analytically Confirmed 25I-NBOMe-Related Death in Washington State.

This case was submitted to the Washington State Patrol Toxicology Laboratory in September 2014. A 15-year-old male went to a party where he ingested 25I-NBOMe and mushrooms. A short time later, he started to vomit and began seizing until he eventually passed out. Resuscitation efforts were made, but were unsuccessful. He was transported to a local hospital, where he died three days later of multi-system organ failure following cardiopulmonary arrest. The hospital admission samples were negative for ethanol and basic drugs and their metabolites. The hospital serum confirmed positive for delta-9-tetrahydrocannabinol (THC) and carboxy-THC at 4.1 and 83 ng/mL, respectively. On the basis of the case history, the hospital blood and urine were sent to NMS Labs for NBOMe and psilocin confirmation. The blood was positive for 25I-NBOMe, and the urine was positive for 25C-, 25H- and 25I-NBOMe, as well as, psilocin. Antemortem and postmortem blood were also sent to AIT Laboratories for NBOMe confirmation. The antemortem blood confirmed positive for 25I-NBOMe with a concentration of 0.76 ng/mL. The manner of death was ruled an accident as a result of combined 25I-NBOMe and psilocin intoxication.

The prevalence of marijuana in suspected impaired driving cases in Washington state.

In December 2012, the possession and private use of limited quantities of marijuana and marijuana products became legal in the state of Washington. At the same time, the state's driving under the influence statutes were amended to include a per se level of 5 ng/mL delta(9)-tetrahydrocannabinol (THC) in whole blood for drivers aged 21 years and older. The aim of this study was to assess the effect of marijuana legalization on the prevalence of marijuana in suspected impaired driving cases. The prevalence of both active THC and its metabolite carboxy-THC detected in such cases pre-legalization was compared with the prevalence post-legalization. In 2009-2012, the average yearly percentage of cases positive for THC and carboxy-THC was 19.1% (range: 18.2-20.2%) and 27.9% (range: 26.3-28.6%), respectively. In 2013, the percentages had significantly increased to 24.9 and 40.0%, respectively (P < 0.05). The median THC concentration over the 5-year period ranged from 5.2 to 6.3 ng/mL, with individual concentrations ranging up to 90 ng/mL. An average of 56% of cases were at or >5 ng/mL over the 5-year period. The prevalence of alcohol and the majority of other drugs in this same population of suspected impaired drivers submitted for testing did not change during this same 5-year period-marijuana was the only drug to show such an increase in frequency. Further, this observed increase remained after the data had been normalized to account for changes in laboratory testing procedures that occurred during this time period. Future studies need be conducted to ascertain whether the observed increase has had any effect on the incidence of crashes, serious injuries and/or traffic fatalities.

XLR-11 and UR-144 in Washington state and state of Alaska driving cases.

The case reports for 18 driving cases positive for the synthetic cannabinoid substances XLR-11 and/or UR-144 are discussed. Eleven of these cases had drug recognition expert evaluations performed. Slurred speech, lack of convergence and body and eyelid tremors were the most consistently noted interview characteristic. Pulse and blood pressure of the subjects were within the expected range. Most of the drivers contacted demonstrated poor driving; however, their performance on the standardized field sobriety tests yielded inconsistent diagnostic information. All cases were negative for other commonly detected drugs that affect the central nervous system, although one case was additionally positive for other synthetic cannabinoids. Of the studied cases, six were positive for only UR-144, whereas eight contained only XLR-11. Four cases were found to have both.

Suspected impaired driving case involving α-pyrrolidinovalerophenone, methylone and ethylone.

This is the first reported case of α-pyrrolidinovalerophenone (α-PVP), methylone and ethylone in a suspected impaired driving case in the state of Washington. An initial traffic stop by law enforcement was made of a driver due to poor navigation of the roadway. The drug recognition expert (DRE) officer observed slurred speech, bloodshot watery eyes, dilated pupils, involuntary muscle movements and an elevated pulse and blood pressure. The DRE deduced that the driver was likely under the influence of central nervous system (CNS) stimulants, specifically 'bath salts'. Routine testing of the blood did not reveal the presence of alcohol or common drugs of abuse. Upon further review of the officer's report and the unconfirmed identification of α-PVP, blood was sent to NMS Labs in Willow Grove, PA, USA for bath salts and stimulant designer drugs testing. Analysis was conducted by liquid chromatography-time-of-flight mass spectrometry with the following results: 63 ng/mL α-PVP, 6.1 ng/mL methylone and positive for ethylone. These results are consistent with the DRE opinion of driving performance being impaired by a CNS stimulant.

Drug-induced hemolytic anemia and thrombocytopenia associated with alterations of cell membrane lipids and acanthocyte formation.

CXCR3 is a chemokine receptor, upregulated upon activation of T cells and expressed on nearly 100% of T cells in sites of inflammation. SCH 900875 is a selective CXCR3 receptor antagonist. Thrombocytopenia and severe hemolytic anemia with acanthocytosis occurred in rats at doses of 75, 100, and 150 mg/kg/day. Massively enlarged spleens corresponded histologically to extramedullary hematopoiesis, macrophages, and hemosiderin pigment and sinus congestion. Phagocytosed erythrocytes and platelets were within splenic macrophages. IgG and/or IgM were not detected on erythrocyte and platelet membranes. Ex vivo increased osmotic fragility of RBCs was observed. Lipid analysis of the RBC membrane revealed modifications in phosphatidylcholine, overall cholesterol, and/or sphingomyelin. Platelets exhibited slender filiform processes on their plasma membranes, analogous to those of acanthocytes. The presence of similar morphological abnormalities in acanthocytes and platelets suggests that possibly similar alterations in the lipid composition of the plasma membrane have taken place in both cell types. This phenotype correlated with alterations in plasma lipids (hypercholesterolemia and low triglycerides) that occurred after SCH 900875 administration, although other factors cannot be excluded. The increased cell destruction was considered triggered by alterations in the lipid profile of the plasma membranes of erythrocytes and platelets, as reflected morphologically.

Prevalence of gabapentin in impaired driving cases in Washington State in 2003-2007.

Gabapentin (Neurontin) is an antiepileptic drug commonly prescribed for pain treatment. In the past 15 years, indications for gabapentin have been increasing even though the complete mechanism of action is unknown. Side effects include somnolence, dizziness, ataxia, nystagmus, and fatigue. This study reviewed all cases positive for gabapentin submitted to the Washington State Toxicology Laboratory between January 2003 and December 2007. The concentrations of gabapentin in blood from impaired driving cases (n = 137) ranged from < 2.0 to 24.7 mg/L with a mean of 8.4 +/- 5.4 mg/L and a median of 7.0 mg/L. The driving population was 50% male with a mean age of 43.0 +/- 10.9 years (range 23-73). Of the cases studied, only 7% were positive for gabapentin alone with the remaining 93% indicative of polydrug use. Drug Recognition Expert reports from four cases in which the only drug detected likely to be causing impairment was gabapentin were examined. These reports demonstrated that subjects may exhibit psychophysical indicators of a central nervous system depressant (e.g., horizontal gaze nystagmus, poor performance on standardized field sobriety tests) with clinical indicators (e.g., dilated pupils, low body temperature, and elevated pulse and blood pressure) that are not consistent with a depressant.

Alterations in phospholipid and fatty acid lipid profiles in primary neocortical cells during oxidant-induced cell injury.

Specific phospholipids and fatty acids altered during oxidant-induced neuronal cell injury were determined using electrospray ionization mass spectrometry (ESI-MS) and ion trapping. The oxidants hydrogen peroxide (H(2)O(2), 0-1000 microM) and tert-butylhydroperoxide (TBHP, 0-400 microM) induced time- and concentration-dependent increases in reactive oxygen species in primary cultures of mouse neocortical cells as determined by 2',7'-dichlorofluorescein diacetate staining and thiobarbituric acid formation. ESI-MS analysis of 26 m/z values, representing 42 different phospholipids, demonstrated that H(2)O(2) and TBHP increased the abundance of phospholipids containing polyunsaturated fatty acids, but had minimal affect on those containing mono- or di-unsaturated fatty acids. These increases correlated to time-dependent increase in 16:1-20:4, 16:0-20:4, 18:1-20:4 and 18:0-20:4 phosphatidylcholine. Oxidant exposure also increased mystric (14:0), palmitic (16:0), and stearic (18:0) acid twofold, oleic acid (18:1) two- to threefold, and arachidonic acid (20:4) fourfold, compared to controls. Increases in arachidonic acid levels occurred prior to increases in the phospholipids, but after increases in ROS, and correlated to increases in oxidized arachidonic acid species, specifically [20:4-OOH]-H(2)O-, 20:4-OH-, and Tri-OH-20:4-arachidonic acid. Treatment of cells with methyl arachidonyl flourophosphonate an inhibitor of Group IV and VI PLA(2), decreased oxidant-induced arachidonic acid release, while bromoenol lactone, an inhibitor of Group VI PLA(2), did not. Collectively, these data identify phospholipids and fatty acids altered during oxidant treatment of neurons and suggest differential roles for Group IV and VI PLA(2) in oxidant-induced neural cell injury.

Involvement of Ca2+-independent phospholipase A2 isoforms in oxidant-induced neural cell death.

This study determined the roles of Ca2+-independent PLA2 (iPLA2) in phospholipid chemistry and oxidant-induced cell death in human astrocytes. A172 cells expressed both cytosolic Group VIA (iPLA2beta) and microsomal Group VIB (iPLA2gamma) PLA2 as determined by activity assays and immunoblot analysis. Inhibition of total iPLA2 activity using racemic bromoenol lactone (BEL, 2.5 microM) decreased the expression of 14:0-16:0 phosphatidylcholine (PtdCho) 15% and increased 18:0-18:1-PtdCho expression 15%. Treatment of cells with the iPLA2gamma specific inhibitor R-BEL decreased 14:0-16:0-PtdCho 35%, 16:0-16:0-PtdCho 15% and induced a 35% increase in 18:0-18:1-PtdCho. In contrast, treatment of cells with the iPLA2beta inhibitor S-BEL did not alter any phospholipid studied. To determine the roles of iPLA2 in oxidant-induced cell death, A172 cells were exposed to hydrogen peroxide (H2O2) or tert-butylhydroperoxide (TBHP); both induced time- and concentration-dependent increases in cell death as assessed by annexin V and propidium iodide staining. Treatment of cells with racemic-BEL alone did not induce cell death. However, pretreatment with BEL prior to H2O2 (500 microM) or TBHP (200 microM) significantly increased necrosis as determined by increases in propidium iodide staining. Treatment with BEL prior to exposure to oxidants accelerated the loss of ATP levels, but not the formation of reactive oxygen species. These data support the hypothesis that iPLA2 mediates oxidant-induced neural cell death and demonstrates differential roles of iPLA2 isoforms in physiological and pathological events.

Differential roles for cytosolic and microsomal Ca2+-independent phospholipase A2 in cell growth and maintenance of phospholipids.

Physiological roles of microsomal (iPLA(2)gamma) and cytosolic (iPLA(2)beta)Ca(2+)-independent phospholipase A(2) were determined in two different epithelial cell models. R- and S-enantiomers of the iPLA(2) inhibitor bromoenol lactone (BEL) were isolated and shown to selectively inhibit iPLA(2gamma) and iPLA(2beta), respectively. The effect of these enantiomers on cell growth was assessed in human embryonic kidney 293 and Caki-1 cells using 3-(4-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT). S-BEL (0-5.0 microM) decreased MTT staining 35% after 24 h compared with control cells, whereas treatment with either R-BEL or R/S-BEL induced 15% decreases. Neither R-BEL nor S-BEL induced cell death as determined by annexin V and propidium iodide staining. Transfection of cells with iPLA(2)beta siRNA reduced MTT staining approximately 35%, whereas transfection of cells with iPLA(2)gamma siRNA only decreased MTT staining 10 to 15% compared with control cells. The effect of iPLA(2)beta and iPLA(2)gamma siRNA on cell number and protein was also determined, and iPLA(2)beta siRNA decreased cell number and protein 25% compared with control cells. In contrast, iPLA(2)gamma siRNA decreased cell number, but not cellular protein, compared with control cells. Selective inhibition of iPLA(2)beta, but not iPLA(2)gamma, decreased several arachidonic acid-containing phospholipids, including 16:1-20:4, 16:0-20:4, 18:1-20:4, and 18:0-20:4 phosphatidylcholine, showing that the ability of iPLA(2)beta inhibitors to decrease cell growth correlates with their ability to decrease arachidonic acid-containing phospholipids. These data show that iPLA(2)beta inhibition results in greater decreases in cell growth and proliferation than iPLA(2)gamma, identifies specific phospholipids whose expressions are differentially regulated by iPLA(2)beta and iPLA(2)gamma, and suggests novel roles for iPLA(2)beta in cell growth.